Monoclonal antibody, hybridoma, their production and use thereof

ABSTRACT

A monoclonal antibody is produced from a cloned hybridoma, and the monoclonal antibody combines specifically with basic fibroblast growth factor (bFGF). Therefore, the monoclonal antibody can be advantageously used for assay reagents on bFGF or for purification of bFGF.

This is a divisional of application Ser. No. 07/915,025 filed Jul. 15,1992, now U.S. Pat. No. 5,478,740 which is a continuation of Ser. No.07/157,453, filed on Feb. 18, 1988, now abandoned.

The present invention relates to monoclonal antibodies which combinespecifically with basic fibroblast growth factor, hybridomas, theirproduction and use thereof.

The basic fibroblast growth factor (also briefly referred to as bFGF, inthe present specification) is a basic polypeptide hormone which issecreted mainly from the pituitary gland and which has a molecularweight of about 17,000. It was first isolated as a factor showing potentgrowth promoting action on fibroblasts such as BALB/C3T3 cells D.Gospodarowicz: Nature, 249, 123 (1974)!. Later, however, it was revealedthat it exhibits growth promoting action on almost all mesoderm-derivedcells D. Gospodarowicz et al.: National Cancer Institute Monograph, 48,109 (1978)!. The neo-vascularizing activity of bFGF, among others,conjointly with its cell growth promoting activity, suggests thepossibility of its use as a therapeutic agent for lesions and burns andas a preventive/therapeutic agent for thrombosis, arteriosclerosis andthe like.

The quantity of naturally occurring human bFGF is very small, andattempts to obtain this factor from human tissues have encounteredserious difficulties arising from various restrictions and limitations.In addition, any method that is easily usable for quantitativedetermination of bFGF has not been established to date. For thesereasons, much remains unknown of basic information which is essentialfor developing bFGF as a drug, such as the properties of bFGF.

Therefore, the development of bFGF as a drug will be facilitated iffurther basic information about bFGF is known, for example, thedistribution of bFGF in vivo and the manner of its production.

In addition, to accurately determine the quantity of bFGF is importantin purifying this protein from gene recombinants. Moreover, it is veryimportant to trace blood FGF concentration in animals which have hadbFGF administered thereto, but blood bFGF cannot be determined by theconventional method using 3T3 cells, due to the mingling of serum in thesample. Usually, the determination of bFGF is achieved by adding bFGF to3T3 cells which have been cultured at reduced serum concentration toattenuate their DNA synthesizing potency, and counting back bFGFconcentration from the degree of recovery of DNA synthesizing potency.However, this method is faulty in that the procedure is delicate anddetermination errors are great, due to the use of cells, and in additionmuch time is needed to obtain results. It is therefore desired that asimple and accurate means of bFGF determination will be developed forthe above-mentioned purpose.

Taking note of the above-mentioned circumstances, the present inventorsmade various investigations to find any practical means of bFGFdetermination, and prepared a monoclonal antibody which combinesspecifically with bFGF and which enables the determination thereof. Thepresent inventors conducted further researches based on thisachievement, and as a result have now developed the present invention.

The present invention provides:

(1) a monoclonal antibody which combines specifically with basicfibroblast growth factor (bFGF), the monoclonal antibody having thecharacteristics:

(a) it has a molecular weight of about 140 to 160 kilodaltons,

(b) it does not cross-react with acidic fibroblast growth factor, and

(c) it belongs to the immunoglobulin class IgM or IgG;

(2) a cloned hybridoma comprising a splenic cell from a mammal immunizedwith bFGF and a homogenic or heterogenic lymphoid cell;

(3) a method for producing a cloned hybridoma comprising a splenic cellfrom a mammal immunized with bFGF and a homogenic or heterogeniclymphoid cell, which comprises subjecting said splenic cell and saidlymphoid cell to cell fusion followed by cloning;

(4) a method for producing a monoclonal antibody which combinesspecifically with bFGF, which comprises growing a cloned hybridomacomprising a splenic cell from a mammal immunized with said factor and ahomogenic or heterogenic lymphoid cell in liquid medium or mammalianabdomen to allow the hybridoma to produce and accumulate the monoclonalantibody;

(5) a method for purifying bFGF, which comprises treating a materialcontaining crude bFGF with the use of the monoclonal antibody defined insaid item (1); and

(6) a method for detecting or measuring bFGF, which comprises using, asantibody, the monoclonal antibody defined in said item (1).

As the bFGF for immunizing mammals, any bFGF can be included, as long asit is a bFGF of a warm-blooded mammal. Its mutein can also be used, soin the present specification "basic fibroblast growth factor (bFGF)" mayinclude its mutein unless otherwise specified.

As representative examples of such mammalian bFGF, mention may be madeof bovine bFGF Proceedings of the National Academy of Sciences, U.S.A.,82, 6507 (1985)! and human bFGF Japanese Patent Application No.241053/1986 which corresponds to European Patent Publication No.237,966; European Molecular Biology Organization (EMBO) Journal, 5, 2523(1986)!.

Polypeptides which includes the amino acid sequence: ##STR1## arepreferred.

More preferably, the polypeptides are represented by the formula:##STR2## wherein X represents Thr or Ser; Y represents Ser when X is Thror Y represents Pro when X is Ser.

For obtaining human bFGF (also briefly referred to as hbFGF), anexpression vector which contains a DNA segment having a base sequenceencoding, for example, the above-mentioned hbFGF protein polypeptide,can be produced, for example, by:

(a) Isolating an RNA encoding hbFGF;

(b) Synthesizing from said RNA a single-stranded complementary DNA(cDNA) and then a double-stranded DNA;

(c) Inserting said complementary DNA into a plasmid;

(d) Transforming a host with the resultant recombinant plasmid;

(e) Cultivating the transformant thus obtained, then isolating theplasmid which contains the desired DNA from the transformant by anappropriate method, for example, the colony hybridization method using aDNA probe;

(f) Cleaving off the desired cloned DNA from said plasmid; and

(g) Inserting said cloned DNA into a vehicle at a site downstream from apromoter.

RNAs encoding hbFGF can be obtained from a wide variety ofhbFGF-producing cells such as human pituitary-derived cells or humanfibroblasts. Such human fibroblasts include WI38 (ATCC No. CCL-75) andIMR90 (ATCC No. CCL-186). Said cell lines WI38 and IMR90 are listed inthe Catalogue of Cell Lines & Hybridomas, 5th edition, 1985, publishedby the American Type Culture Collection.

By inserting the expression vector thus obtained into an appropriatehost (e.g., Escherichia coli, Bacillus subitlis, yeasts, animal cells),and cultivating the obtained transformant in a medium, human bFGF can beproduced.

The muteins in the present invention essentially have the amino acidsequence of the original peptide or protein; but variations include anaddition of amino acid(s), deletion of constituent amino acid(s) andsubstitution of constituent amino acid(s) by other amino acid(s).

Such addition of amino acid includes addition of at least one aminoacid. Such deletion of constituent amino acid includes deletion of atleast one bFGF-constituent amino acid. Such substitution of constituentamino acid by other amino acids includes substitution of at least onebFGF-constituent amino acid by other amino acid.

The at least one amino acid in the mutein which has at least one aminoacid added to bFGF excludes methionine deriving from initiation codonused for peptide expression and signal peptide.

The number of added amino acids is at least 1, but it may be any one, aslong as bFGF characteristics are not lost. Preferable amino acids shouldinclude some or all of the amino acid sequences of proteins which havehomology with bFGF and which exhibit activities similar to those ofbFGF.

As for the number of deleted bFGF-constituent amino acids in the presentmutein which lacks at least one bFGF-constituent amino acid, it may beany one, as long as any characteristic of bFGF is not lost.

Examples of such deleted constituent amino acids include: the deletionof amino acids from amino terminal or carboxyl terminal; the deletion ofthe 10 residues in the amino terminal of human bFGF: ##STR3## the 14residues in the amino terminal of human bFGF: ##STR4## the 41 residuesin the amino terminal of human bFGF: ##STR5## or the 61 residues in thecarboxyl terminal of human bFGF: ##STR6##

As for the number of bFGF-constituent amino acids that may besubstituted by other amino acids before substitution in mutein is lost,it may be any number, as long as any characteristic of bFGF is not lost.

As examples of constituent amino acids before substitution, mention maybe made of cysteine and other amino acids but cysteine is preferable. Asthe constituent amino acid other than cysteine which may be substitutedfor, examples include but are not limited to aspartic acid, arginine,glycine, serine, valine and so forth.

When the constituent amino acid before substitution is cysteine, thesubstituted amino acids are preferably, for example, neutral aminoacids. As specific examples of such neutral amino acids, mention may bemade of glycine, valine, alanine, leucine, isoleucine, tyrosine,phenylalanine, histidine, tryptophan, serine, threonine and methionine.Serine and threonine are preferable.

When the constituent amino acid before substitution is other thancysteine, the substituted amino acids are selected from amino acidswhich are different from the amino acid before substitution in aproperty such as hydrophilicity, hydrophobicity or electric charge.

When the constituent amino acid before substitution is aspartic acid,examples of the substituted amino acids include asparagine, threonine,valine, phenylalanine and arginine; asparagine and arginine arepreferable.

When the constituent amino acid before substitution is arginine,examples of the substituted amino acids include glutamine, threonine,leucine, phenylalanine and aspartic acid; glutamine is preferable.

When the constituent amino acid before substitution is glycine, examplesof the substituted amino acids include threonine, leucine,phenylalanine, serine, glutamic acid, and arginine; threonine ispreferable.

When the constituent amino acid before substitution is serine, examplesof the substituted amino acids include methionine, alanine, leucine,cysteine, glutamine, arginine and aspartic acid; methionine ispreferable.

When the constituent amino acid before substitution is valine, examplesof the substituted amino acids include serine, leucine, proline,glycine, lysine, and aspartic acid; serine is preferable.

The constituent amino acid before substitution are preferably asparticacid, arginine, glycine, serine and valine.

The substituted amino acid is preferably asparagine, glutamine,arginine, threonine, methionine, serine, and leucine.

The embodiment on the substitution in the mutein wherein there is asubstitution of serine for cysteine (i.e. cysteine is replaced byserine) is most preferred.

In said substitution, there may be at least 2 substitutions and two orthree substitutions are preferred.

The muteins in the present invention include combination of 2 or 3 ofthe above-mentioned additions, deletions and substitutions.

For producing said muteins, site-directed mutagenesis is carried out.This technique is well-known, and is described in Lather, R. F. andLecoq, J. P., Genetic Engineering, Academic Press (1983), pp. 31-50.Mutagenesis which is directed to oligonucleotide is described in Smith,M. and Gillam, S., Genetic Engineering: Principles and Methods, PrenamPress (1981), vol. 3, pp. 1-32.

For producing a structural gene encoding said mutein, for example:

(a) a single-stranded DNA consisting of a single strand of a structuralgene of bFGF is hybridized with a mutant oligonecleotide primer (theabove-mentioned primer is complementary to a region including thecysteine codon to be replaced by this single strand or, as the case maybe, an antisense triplet which forms a pair with this codon, except thatdiscrepancies with codons for amino acid coding other than the relevantcodon or, as the case may be, with antisense triplets are permitted.),

(b) the primer is elongated by DNA polymerase to allow it to form amutational heteroduplex, and

(c) this mutational heteroduplex is replicated.

The phage DNA carrying the mutated gene is then isolated and insertedinto a plasmid.

The plasmid thus obtained is used to transform an appropriate host (asmentioned above), and the resulting transformant is cultured in amedium, whereby mutein can be produced.

In immunizing said bFGF, the bFGF may be prepared in a complex form witha carrier protein before use.

Such carrier proteins include, for example, Freund's complete adjuvant(Difco Laboratories).

When a carrier protein complex is used, the coupling ratio of carrierprotein to bFGF is about 5 to 30 times (carrier/bFGF, ratio by weight).It is preferable that the ratio be about 15 to 20 times.

For coupling between hapten and carrier, various condensing agents canbe used, but glutaraldehyde, carbodiimide, etc. are preferably used.

In immunizing mammals by means of bFGF or a protein complex, laboratoryanimals such as sheep, goats, rabbits, guinea pigs, rats and mice may beused, and rats and mice, especially mice, are preferred for obtainingmonoclonal antibodies. As to the method of immunization, immunization ispossible via any route such as subcutaneous, intraperitoneal,intravenous, intramuscular or intracutaneous injection, but it ispreferable that the immunogen be injected mainly subcutaneously,intraperitoneally or intravenously (in particular, subcutaneously). Inaddition, immunizing interval, immunizing dose, etc. are also highlyvariable, allowing various methods to be carried out; the method inwhich immunization is conducted about 2 to 6 times at intervals of 2weeks, and splenic cells taken out about 1 to 5 times, preferably about2 to 4 days after the final immunization are used, for example, iscommonly used. It is desirable that an immunizing dose of more thanabout 0.1 μg, preferably about 10 to 300 μg for each mouse, calculatedon the peptide amount basis, be used in each injection. It is alsodesirable that a fusion experiment using a splenic cell be conductedafter certification of increase in blood antibody titer by local bloodsampling prior to excision of the spleen.

In the above-mentioned cell fusion of a splenic cell with a lymphoidcell, an excised mouse splenic cell, for example, is fused with anappropriate homogenic or heterogenic (preferably homogenic) lymphoidcell line having a marker such as hypoxanthine-guaninephosphoribosyltransferase deficiency (HGPRT⁻) or thymidine kinasedeficiency (TK⁻). As the lymphoid cell line, myeloma cell is preferred,and the myeloma cell there is mentioned myeloma P3-X63-Ag. 8UI (Ichimoriet al.: Journal of Immunological Methods, 80, 55 (1985)!. This fusioncan be executed via e.g. the method developed by Kohler and MilsteinNature, 256, 495 (1975)!. For example, myeloma cells and splenic cells,in an about 1:5 ratio, are suspended in a medium prepared by mixingtogether Iskov medium and Ham F-12 medium in a 1:1 ratio (hereinafterreferred to as IH medium), and a fusing agent such as Sendai virus orpolyethylene glycol (PEG) is used. Of course, dimethyl sulfoxide (DMSO)and/or other fusion promoters can also be added. The following arenormally used: a degree of polymerization for the PEG of about 1000 to6000, a treating time of about 0.5 to 30 minutes and a PEG concentrationof about 10 to 80%. Efficient fusion can be achieved by about 4 to 10minutes of PEG 6000 treatment at an about 35 to 55% concentration. Thefused cells can be selectively grown using thehypoxanthine-aminopterin-thymidine medium HAT medium; Nature, 256, 495(1975)! or the like.

The culture supernatant of grown cells can be subjected to screening forthe production of the desired antibody, and screening for antibody titercan be conducted as follows: In this case, the culture supernatant canfirst be assayed for the production of antibody to an immunized peptideby a method such as the radioimmunoassay (RIA) method or enzymeimmunoassay (EIA) method. These methods are also widely modifiable. Asan example of preferred method of assay, a method using EIA is describedbelow. To a carrier such as cellulose beads the rabbit anti-mouseimmunoglobulin antibody, for example, is beforehand coupled inaccordance with a routine method, and the culture supernatant to beassayed and mouse serum are added thereto, and reaction is carried outat constant temperature (which means about 4° to 40° C.; this definitionalso applied hereinafter) for the specified time. After the reactionproduct is well washed, a peptide labeled with enzyme (prepared bycoupling of an enzyme and a peptide in accordance with a routine method,followed by purification) is added, and reaction is carried out atconstant temperature for the specified time. After the reaction productis well washed, an enzyme substrate is added, and reaction is carriedout at constant temperature for the specified time, whereafter theresulting chromogenic substance can be assayed by absorptiometry orfluorometry.

It is desirable that the cells, which showed proliferation in theselective medium and secreted antibodies which combined with peptideused for the immunization, were subjected to cloning by limitingdilution analysis etc. The supernatant of the cloned cells is subjectedto screening in the same manner as above to increase cells in the cellshigh in antibody titer, whereby monoclonal antibody producing hybridomaclones showing reactivity to the immunized peptide are obtained.

The hybridoma thus cloned is grown in liquid medium, for example, amedium prepared by adding about 0.1 to 40% bovine serum to RPMI-1640Moore, G. E. et al.; Journal of American Medical Association, 199, 549(1967)!. Specifically, said monoclonal antibody can be obtained from themedium cultured for about 2 to 10 days, preferably about 3 to 5 days.The antibody can also be obtained from ascites fluids of mice which areintraperitoneally inoculated the hybridoma. For this purpose, in thecase of mice, for example, about 1×10⁴ to 1×10⁷, preferably about 5×10⁵to 2×10⁶ hybridoma cells are intraperitoneally inoculated to a mouse ofBALB/c or similar strain, previously inoculated with mineral oil etc.,and about 7 to 20 days later, preferably about 10 to 14 days laterascites fluid is collected. The antibody produced and accumulated in theascites is subjected to, for example, ammonium sulfate fractionation andDEAE-cellulose column chromatography, whereby the desired monoclonalantibody can easily be isolated as a pure immunoglobulin.

A monoclonal antibody which combines specifically with bFGF is thusobtained.

The monoclonal antibody of the present invention combines specificallywith the immunogen peptide bFGF. The monoclonal antibody of the presentinvention may also combine with a bFGF other than the immunogen peptide.The monoclonal antibody of the present invention is a monoclonalantibody to the immunogen peptide bFGF or its mutein. As the presentmonoclonal antibody reacts with only bFGF (and its mutein), the presentmonoclonal antibody combines specifically with bFGF.

As shown in Example 3 below, when human bFGF is used as an immunogen, amonoclonal antibody belonging to the immunoglobulin class IgM isobtained in some cases.

Since combining specifically with bFGF, the monoclonal antibody of thepresent invention is very useful as a reagent for bFGF assay. It alsofacilitates bFGF assay in living organs and tissues, so it is veryuseful also in obtaining basic information about bFGF (e.g.,distribution in vivo). In the detection procedure on the bFGF in livingorgans and tissues, the measuring by EIA method or fluorescent antibodytechnique are generally employed. In order to measure the amount in theliving organs and tissues, it is always employed Western blotting methodon protein. In this method, a crude extract or partial purified sampleof the extract is subjected to electrophoresis with acylamide gel,transferring to membrane filter, and then detection with HRP-anti bFGFantibody.

In addition, it is thought that some cancer cells produce bFGF bythemselves to continue their proliferation on the basis of the bFGF.When anti-bFGF antibody is allowed to act on such cancer, theproliferation-promoting bFGF is neutralized, and the antibody isexpected to exhibit cancer cell proliferation inhibition, that is, toact as an anticancer substance. In addition, the antibody can be used todetermine the bFGF in bFGF-producing cancer, so it can also be appliedto cancer diagnostic reagents. Moreover, based on the avidity of thesaid antibody to bFGF, an antibody affinity column can be prepared touse the antibody as a reagent for bFGF purification.

As the EIA method and RIA method for detecting or measuring bFGF, thereare mentioned the following procedure.

For example, a purified antibody is fixed on 96 wells plastic plate(e.g. Immunoplate, Nunc, Denmark) at about 0.1 to 10 μg/well, glassbeads, plastic beads. The fixation is carried out at about 4° C. forovernight, or at room temperature for about 0.4 to 4 hours, in case ofplastic. In case of glass beads, the fixation is carried out inaccordance with the method described in Proc. Natl. Acad. Sci. U.S.A.,80, 3513-3516 (1983). Thus obtained plate or beads to which the antibodyhas been fixed is subjected to adsorption reaction with the antigen bFGF(or its mutein). The adsorption reaction is generally carried out atroom temperature for about 0.2 to 2 hours, preferably about 4° C.overnight. After the antigen-antibody reaction, adsorption reaction iscarried out by adding an antibody which has been labeled with an enzymein case of EIA or labeled with a radioisotope in case of RIA. As theenzyme to label an antibody, there are exemplified by horse radishperoxidase (HRP), alkaline phosphatase. As the examples of radioisotopelabeling, there are mentioned ¹²⁵ I.

In case of EIA, a substrate such as 2,2'-adino-di 3-ethylbenzothiazolinesulfonate (6)! is employed for coloring when HRP is employed as alabeling enzyme.

In case of RIA, the radioactivity is measured by scintillation counter.The measuring the bFGF is carried out by comparing the absorbancy orradioactivity with those of the known amount of bFGF.

As the EIA method, there are mentioned sandwich EIA method, competitiveEIA method, indirect EIA method. In the sandwich method, two antibodiesare bound by mediating the antigen, bFGF. In the competitive EIA method,an antibody is fixed to a carrier, antigen bFGF which has been combinedwith a labeling enzyme or radioisotope, and a sample, so as to reactcompetitively, and then measuring the amount of labeled antigen. In thiscompetitive EIA method, the reaction conditions, and measuring theamount of bound antibody to antigen are the same as the sandwich EIAmethod. As the indirect EIA method, a sample and an antibody (which isnot fixed) are reacted with each other, the unbound antibody is measuredby a plate to which the antigen is fixed and an anti-mouse antibody. Inthis indirect EIA method, the reaction conditions and measuring methodare those as mentioned above.

For the purpose of bFGF purification, efficient purification can beachieved by, for example, the method in which the purified relevantantibody, after coupling with an appropriate carrier such as theactivated agarose bead in accordance with a routine method, is packed ina column, the crude sample containing bFGF such as culture supernatantor disrupted bacterial cells is adsorbed to the antibody affinitycolumn, and the column is washed, whereafter bFGF is eluted with achaotropic reagent such as KSCN (potassium thiocyanate) or under suchslightly acidic conditions that bFGF is never inactivated.

The preparation of antibody column is carried out by coupling themonoclonal antibody of the present invention, purified from, forexample, ascites fluid inoculated with hybridoma, with an appropriatecarrier, by the method as follows:

Any carrier can be used, as long as it ensures the efficient adsorptionspecifically of bFGF after coupling, and enables the appropriate elutionof bFGF after the adsorption; as the carrier there are mentioned polymerof agarose, cellulose or acrylamide, and as the carrier, for example,polyacrylamide gel beads activated so that primary amino group inprotein is easily combined with, such as Affi-Gel-10 is used asappropriate in the manner as described below. The reaction betweenAffi-Gel-10 and antibodies is carried out in a buffer such as a solutionof about 0.001 to 1M, preferably about 0.1M bicarbonate. As to reactionconditions, the reaction can be carried out at about 0° to 20° C. forabout 10 to 24 hours at any pH level, but about 4° C., about 4 hours,and pH about 3 to 10 is preferably used for reaction conditions. Sincemore antibodies are adsorbed as the amount of antibodies per 1 m ofAffi-Gel-10 increases, as long as the amount is less than about 50 mg;therefore, any quantitative ratio between Affi-Gel-10 and antibodies tobe mixed together can be chosen within this range, but about 10 to 30 mgof antibodies is used as appropriate, considering binding efficiency andpurifying efficiency in affinity column chromatography. Theseantibody-carrier conjugates can be used for an antibody colum by packingin an appropriate column after blocking the remaining unreacted activegroups by a method such as the method in which the conjugates, afterbeing well washed with the buffer used for the reaction, are keptstanding several days, or the method in which a final concentration ofabout 0.05 to 0.1M of a compound having a primary amino group such asethanolamine hydrochloric acid or glycine is added and reaction iscarried out at about 4° C. for about 1 to 4 hours, or about 1 to 5% ofprotein such as bovine serum albumin (BSA) is added and reaction iscarried out at 4° C. overnight.

In purification using the above-mentioned antibody column, for example asample containing bFGF is dissolved in a buffer such as a phosphatebuffer or a Tris hydrochloric acid and is adsorbed to the antibodycolumn. Thereafter, the column is washed with the same buffer, and bFGFis then eluted. As eluents, there can be used slightly acidic solutionssuch as acetic acid solutions, solutions containing polyethylene glycol,solutions containing peptide which is more likely to combine withantibodies than the sample, high concentration salt solutions, andsolutions prepared by combining these, and those which do notconsiderably accelerate the decomposition of bFGF are preferred.

The column effluent is neutralized with a buffer by a routine method.Where necessary, a purifying procedure using the above antibody columncan be again carried out.

In this way, substantially pure bFGF mutein protein can be obtained. Thesubstantially pure bFGF mutated protein according to this inventionincludes products whose bFGF mutated protein content is not less than90% (w/w) and, more preferably, products whose bFGF mutein content isnot less than 95% (w/w).

The bFGF solution thus obtained are subjected to dialysis, and, ifnecessary, can be made into a powder by lyophilization. In thelyophilization, there may be added stabilizers such as sorbitol,mannitol, dextrose, maltose and glycerol.

The hbFGF thus obtained possesses growth promoting activity offibroblast cells and endothelial cells and angiogenic activity, and itstoxicity is low; therefore, the hbFGF can be used as a cure promoter forburns, wounds, postoperative tissues, etc., or as a therapeutic agentfor thrombosis, arteriosclerosis, etc. which is based on itsneovascularizing effect. Furthermore, it can be used as a reagent forpromoting cell cultivation.

For its pharmaceutical use, the hbFGF can be safely administered towarm-blooded mammals (e.g. humans, mice, rats, hamsters, rabbits, dogs,cats) parenterally or orally either per se in a powder form or in theform of pharmaceutical compositions (e.g., injection, tablet, capsule,solution, ointment) made up together with pharmacologically acceptablecarriers, excipients and/or diluents.

Injectable preparations can be produced by a conventional method using,for example, physiological saline or an aqueous solution containingglucose and/or other adjuvant or adjuvants. Tablets, capsules and otherpharmaceutical compositions can also be prepared in accordance with aconventional method. When prepared for use as a pharmaceutical, careshould be taken that aseptic conditions are used and that the resultantproduct is sterile, low in pyrogens and endotoxins.

When used for the above pharmaceutical purposes, the hbFGF isadministered, for example, to the above warm-blooded mammals in anappropriate amount selected from the range of from about 1 ng to 100μg/kg body weight a day according to the route of administration,symptoms, etc.

When used as a reagent for promoting cell cultivation, the hbFGF isadded to the medium preferably in an amount of 0.01 to 10 μg, morepreferably 0.1 to 10 μg per liter of medium.

The mutein of hbFGF can also be used as with the above hbFGF.

Thus, since combining specifically with bFGF, the monoclonal antibodiesof the present invention can be advantageously used for bFGF assayreagents and for purifying bFGF.

In addition, of the monoclonal antibodies of the present invention,those which are high in antibody valency are advantageous in that, whenthey are used as bFGF assay reagents, the amount of other reagentsprepared at the time of use, for example, antiserum, is saved.Furthermore, bFGF purification to higher degree can be achieved by theuse thereof.

In the specification and drawings of the present invention, theabbreviations used for bases, amino acids and so on are thoserecommended by the IUPAC-IUB Commission on Biochemical Nomenclature orthose conventionally used in the art. Examples thereof are given below.Amino acids for which optical isomerism is possible are, unlessotherwise specified, in the L form.

DNA: Deoxyribonucleic acid

cDNA: Complementary deoxyribonucleic acid

A: Adenine

T: Thymine

G: Guanine

C: Cytosine

RNA: Ribonucleic acid

dATP: Deoxyadenosine triphosphate

dTTP: Deoxythymidine triphosphate

dGTP: Deoxyguanosine triphosphate

dCTP: Deoxycytidine triphosphate

ATP: Adenosine triphosphate

Tdr: Thymidine

EDTA: Ethylenediaminetetraacetic acid

SDS: Sodium dodecyl sulfate

Gly: Glycine

Ala: Alanine

Val: Valine

Leu: Leucine

Ile: Isoleucine

Ser: Serine

Thr: Threonine

Cys: Cysteine

Met: Methionine

Glu: Glutamic acid

Asp: Aspartic acid

Lys: Lysine

Arg: Arginine

His: Histidine

Phe: Phenylalanine

Tyr: Tyrosine

Trp: Tryptophan

Pro: Proline

Asn: Asparagine

Gln: Glutamine

In Reference Examples mentioned below, the human bFGF constituent aminoacids shall be numbered according to the rule, in which Met added to theN-terminal of the peptide having Thr for X and Ser for Y in theabove-mentioned amino acid sequence (II), said Met is numbered as thefirst.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the human bFGF-encoding base sequence as determined inReference Example 1 and the amino acid sequence deducible from said basesequence.

FIG. 2 shows the polyacrylamide gel electrophoresis patterns onimmunoprecipitation as obtained in Example 5.

FIG. 3 shows the antibody valencies as obtained in Example 6.

FIG. 4 shows the results of competitive inhibition experiment to MoAb12by the various peptides as obtained in Example 7.

FIG. 5 shows the results of competitive inhibition experiment to MoAb52by the various peptides as obtained in Example 7.

FIG. 6 shows the results of competitive inhibition experiment to MoAb78by the various peptides as obtained in Example 7.

FIG. 7 shows the results of competitive inhibition experiment to MoAb98by the various peptides, as obtained in Example 7.

FIG. 8 shows the results of competitive inhibition experiment on bFGFmutein of MoAb52 as obtained in Example 7.

FIG. 9 shows the results of competitive inhibition experiment on bFGFmutein of MoAb as obtained in Example 7.

FIG. 10 shows the pattern of polyacrylamide gel electrophoresis asobtained in Example 9.

FIG. 11 shows the quantification of bFGF by EIA using MoAb52 andHRP-MoAb78.

FIG. 12 shows the quantification of bFGF by EIA using MoAb98 andHRP-MoAb78.

FIG. 13 shows the detection of bFGF by Western blotting method usingmonoclonal antibody 78 as obtained in Example 12.

The mouse HbF99 cell, mouse HbF161 cell and mouse HbF165 cell obtainedin Example 2 (3) to be described later have been deposited at Institutefor Fermentation, Osaka (IFO), Japan since Jan. 28, 1987 respectivelyunder the following accession numbers:

Mouse HbF99 cell: IFO 50122

Mouse HbF161 cell: IFO 50123

Mouse HbF165 cell: IFO 50124

The mouse hybridomas HbF12, HbF52, HbF78, and HbF98 obtained in Example4 to be described later have respectively been deposited at the IFOsince Aug. 17, 1987 under the following accession numbers:

Mouse HbF12 cell: IFO 50142

Mouse HbF52 cell: IFO 50143

Mouse HbF78 cell: IFO 50144

Mouse HbF98 cell: IFO 50145

The following transformants which were produced in the ReferenceExamples mentioned below were deposited at IFO and at the FermentationResearch Institute, Agency of Industrial Science and Technology,Ministry of International Trade and Industry (FRI), Japan under theaccession numbers on the deposit dates shown in Table 1 (The depositdates are indicated in parentheses.). As to the deposit number of FRI,FERM BP number denotes the number of deposit under the Budapest Treaty;and in case both FERM P number and FERM BP number are described, itshows that the deposit under the number of FERM P has been converted tothe deposit under Budapest Treaty and the transformants have been storedat FRI under the number of FERM BP.

                  TABLE 1    ______________________________________    Transformants             IFO         FRI    ______________________________________    E. coli K12             IFO 14494   FERM P-8726 FERM BP-1280    DHI/pTB 627             (March 13, 1986)                         (April 2, 1986)     Reference    Example 1!    E. coli K12             IFO 14532   FERM P-8918 FERM BP-1281    MM294/pTB             (August 11, 1986)                         (August 21, 1986)    669  Reference    Example 2!    E. coli DH1/             IFO 14575   FERM P-9216 FERM BP-1641    pTB 739  (February 18,                         (February 25,     Reference             1987)       1987)    Example 5!    E. coli DH1/             IFO 14584   FERM P-9307 FERM BP-1642    pTB 742  (March 24, 1987)                         (March 30, 1987)     Reference    Example 6!    E. coli DH1/             IFO 14585   FERM P-9308 FERM BP-1643    pTB 743  (March 24, 1987)                         (March 30, 1987)     Reference    Example 7!    E. coli DH1/             IFO 14586   FERM P-9309 FERM BP-1644    pTB 744  (March 24, 1987)                         (March 30, 1987)     Reference    Example 8!    E. coli  IFO 14613   FERM P-9409 FERM BP-1645    MM294/pTB             (May 27, 1987)                         (June 11, 1987)    762  Reference    Example 10!    E. coli  IFO 14700               FERM BP-1660    MM294/pTB             (January 14,            (January 20,    795  Reference             1988)                   1988)    Example 12!    E. coli  IFO 14701               FERM BP-1661    MM294/pTB             (January 14,            (January 20,    796  Reference             1988)                   1988)    Example 13!    E. coli  IFO 14702               FERM BP-1662    MM294/pTB             (January 14,            (January 20,    797  Reference             1988)                   1988)    Example 14!    ______________________________________

EXAMPLES

The present invention will now be illustrated in more detail by means ofthe following working examples, but the present invention is neverlimited thereby.

Reference Example 1

(Construction of a plasmid containing an hbFGF-encoding gene)

(1) Isolation of cDNA-containing plasmid:

A cDNA library with Escherichia coli x1776 as the host as produced byinserting cDNA synthesized from human foreskin derived primary culturecell mRNA into the pCD vector Okayama et al.: Molecular and CellularBiology, 3, 280 (1983)! was provided by Dr. Okayama at the NationalInstitute of Child Health and Human Development, Bethesda, U.S.A. Theplasmid DNA was extracted from this cDNA library by the alkalineextraction method Birnboim, H. C. and Doly, J.: Nucleic Acids Research,1, 1513 (1979)! and Escherichia coli DH1 was infected with this DNA. AcDNA library comprising about 2×10⁶ clones was thus produced withEscherichia coli DH1 as the host.

The above cDNA library with Escherichia coli DH1 used therein was platedon 10 pieces of nitrocellulose filter (Millipore's HATF filter) in anamount of about 5×10⁴ clones per filter. Using these filters as masterfilters, 20 replica filters were prepared in 10 pairs corresponding tothe master filters. Escherichia coli cells on these replica filters werelysed with a 0.5N NaOH solution and plasmid DNAs exposed and denaturedwere immobilized on the filters Grunstein, M. & Hogness, D. S.: Proc.Natl. Acad. Sci. U.S.A., 72, 3961(1975)!.

Based on the amino acid sequence of bovine basic fibroblast growthfactor as reported by F. Esch et al. Proc. Natl. Acad. Sci. U.S.A., 82,6507 (1985)!, base sequences corresponding to two amino acid sequencescovering amino acid Nos. 13-20 (Pro-Pro-Gly-His-Phe-Lys-Asp-Pro) andamino acid Nos. 89-96 (Thr-Asp-Glu-Cys-Phe-Phe-Phe-Glu), respectivelywere chemically synthesized. (In some codons, the third letter wasselected arbitrarily. Thus, the base sequences synthesized were 5'GG^(A) /_(G) TC^(T) /_(C) TT^(A) /_(G) AA^(A) /G TGGCCAGGAGG and 5'TC^(A) /_(G) AA^(A) /_(G) AA^(A) /_(G) AA^(A) /_(G) CA^(T) /_(C)TCGTCGGT, with each underlined base being the one selected.) Theseoligonucleotides were labeled with ³² P at the 5' end by treating saidoligonucleotides in 50 μl of reaction mixture 0.1 μg of oligonucleotide,50 mM Tris-HCl, pH 8.0, 10 mM MgCl₂, 10 mM mercaptoethanol, 50 μCi γ-³²P ATP (>5,000 Ci/mmole), 3 units of T4 polynucleotide kinase (TakaraShuzo, Japan)! at 37° C. for 1 hour.

The thus-labeled oligonucleotides of the above two kinds wereindividually hybridized as probes with the replica filters. Thehybridization reaction was conducted in 10 ml of a 100 μg/ml denaturedsalmon sperm DNA solution containing 10 μCi of probe in 5×SSPE 180 mMNaCl, 10 mM NaH₂ PO₄, 1 mM EDTA (pH 7.4)! and 5×Denhardt's with 0.1% SDSat 35° C. for 16 hours. After reaction, the filters were washed with a0.1% SDS solution in 5×SSC 0.15M NaCl, 0.015M sodium citrate! threetimes each at room temperature for 30 minutes and then two times each at45° C. for 30 minutes T. Maniatis et al.: "Molecular Cloning", ColdSpring Harbor Laboratory, p. 309 (1982)!.

Radioautograms were taken for the washed filters. A bacterial straincapable of reacting with the both kinds of probe was searched for bysuperposing the radioautograms for each pair of replica filters. In thismanner, a strain Escherichia coli K12 DH1/pTB627 (IFO 14494, FERMBP-1280)! capable of reacting with the two kinds of probe was obtainedfrom among 5×10⁵ colonies.

(2) The plasmid DNA (pTB627) was extracted from the strain obtainedabove in (1) Escherichia coli K12 DH1/pTB627 (IFO 14494, FERM BP-1280)!by the alkaline extraction method Nucleic Acids Research, 1, 1513(1979)! and purified.

(3) Then, the base sequence of the cDNA portion encoding hbFGF wasdetermined by the dideoxynucleotide synthetic chain termination methodJ. Messing et al.: Nucleic Acids Research, 9, 309 (1981)!. The aminoacid sequence deduced from said base sequence is shown in FIG. 1.

Reference Example 2

(Expression of hbFGF-encoding gene in Escherichia coli)

Construction of hbFGF expression plasmid pTB669:

The plasmid pTB627 obtained in Reference Example 1 (2) mentioned aboveand containing the hbFGF cDNA was cleaved with the restriction enzymesAvaI and BalI, whereby a 0.44 kb DNA fragment containing thehbFGF-encoding region was obtained. A BglII linker, pCAGATCTG, wasligated with this DNA fragment at its BalI cleavage site (blunt end) byT4 DNA ligase, and a 0.44 Kb AvaI-BglII DNA fragment was isolated. T4DNA ligase was allowed to act on this 0.44 Kb AvaI-BglII fragment tothereby cause ligation between the BglII cleavage sites. Then, DNApolymerase (Klenow fragment) reaction was carried out in the presence ofdXTPs to render the AvaI cleavage sites blunt. This DNA fragment wasligated with phosphorylated synthetic oligonucleotides, ^(5')AATTCTATGCCAGCATTGC^(3') and ^(5') GCAATGCTGGCATAG^(3'), in the presenceof T4 DNA ligase. An about 0.46 kb DNA fragment was then prepared bycleavage with EcoRI-BglII. Separately, the trp promoter-containingplasmid ptrp781 Kurokawa, T. et al.: Nucleic Acids Research, 11,3077-3085 (1983)! was cleaved with PstI and rendered blunt-ended by T4DNA polymerase reaction. The BglII linker pCAGATCTG was joined to theabove cleavage product at the blunt ends thereof by T4 DNA ligasereaction; then the ligation product was cleaved with EcoRI-BglII and anabout 3.2 kb DNA fragment containing the trp promoter, the tetracyclineresistance gene and the plasmid replication origin was isolated. This3.2 kb DNA fragment was ligated with the above-mentioned 0.46 kbEcoRI-BglII DNA fragment containing the hbFGF-encoding gene region by T4DNA ligase reaction, whereby an hbFGF expression plasmid, pTB669, wasconstructed.

This plasmid pTB669 was used to transform Escherichia coli DH1 to give atransformant carrying the plasmid pTB669, namely Escherichia coliDH1/pTB669.

pTB669 was also used in the same manner to transform the Escherichiacoli strains K12MM294 and C600 to give Escherichia coli K12 MM294/pTB669(IFO 14532, FERM BP-1281) and E. coli C600/pTB669, respectively.

Reference Example 3

(Purification of human basic fibroblast growth factor (hbFGF))

Escherichia coli K12MM294/pTB669 (IFO 14532, FERM BP-1281) as obtainedin Reference Example 2 was cultivated in M9 medium Maniatis, T. et al.:Molecular Cloning (1982), A Laboratory Manual, Cold Spring HarborLaboratory, U.S.A.! containing 1% glucose, 0.4% casamino acid and 8μg/ml tetracycline. When the Klett value reached about 200,3-β-indolylacrylic acid was added to 25 μg/ml, and the cultivation wascontinued for 4 more hours. Thereafter, cells were harvested andsuspended in one twentieth volume of 10% sucrose solution in 20 mMTris-HCl, pH 7.6. To this suspension were added phenylmethylsulfonylfluoride (PMSF) to 1 mM (final concentration), EDTA to 10 mM, NaCl to0.1M, spermidine hydrochloride to 10 mM and lysozyme to 100 μg/ml. Afterallowing to stand at 0° C. for 45 minutes, the whole mixture wassonicated for 30 seconds. The sonication product was centrifuged at18,000 rpm (Sorvall centrifuge, SS 34 rotor, U.S.A.) for 30 minutes togive a supernatant, which was used as the cell extract.

A 25-ml portion of this extract (as prepared from 500 ml of culturebroth) was passed through a DEAE-cellulose (DE52, Whatman, England)column (diameter 2×10 cm) equilibrated with 0.2M NaCl solution in 20 mMTris-HCl, pH 7.6 to thereby remove nucleic acid components in theextract. The effluent from the column and the column washings resultantfrom washing with 0.2M NaCl solution in 20 mM Tris-HCl, pH 7.6 werecollected and combined (DEAE effluent fraction 44 ml).

A 14-ml portion of this fraction was applied to a high performanceliquid chromatograph (Gilson, France) equipped with a heparin columnShodex AF-pak HR-894 (8 mm ID×5 cm, Showa Denko, Japan). The column waswashed with 20 mM Tris-HCl, pH 7.6. Thereafter, elution was performed ona linear gradient of 0.5-2M NaCl in 20 mM Tris-HCl buffer, pH 7.6,(eluent volume 60 ml, flow rate 1.0 ml/min).

The hbFGF eluted by this procedure showed a single band inSDS-polyacrylamide gel electrophoresis, and it was thus found to besufficiently purified and suitable for use as an antigen. The assay ofhbFGF was conducted using the following conditions.

A Nunc 96-well microtiter plate (flat bottomed) was sown with mouseBALB/c3T3 cells (2×10³ cells per well) with DMEM medium containing 5%calf serum (0.2 ml per well) and the cells were cultured. Next day, themedium was replaced with DMEM medium containing 0.5% calf serum. After 3days of cultivation, dilutions of the cell extract as prepared by serial5-fold dilution with DME medium containing 0.5% BSA were added in anamount of 10 μl per well. After 20 hours of continued cultivation, 2 μlof ³ H-Tdr (5 Ci/mmol, 0.5 mCi/ml RCC Amersham) was added to each well.Six hours later, cells in each well were scraped off by treatment withphosphate buffer (PBS) containing 0.2% trypsin and 0.02% EDTA andcollected on a glass filter using a Titertek cell harvester, and thequantity of ³ H-Tdr taken up by the cells was measured using ascintillation counter.

Reference Example 4

(Production of recombinant DNA having mutein-encoding base sequence)

(1) Cloning of human bFGF gene M13 vector

The plasmid pTB669 obtained in Reference Example 2 was digested with therestriction enzymes EcoRI and BamHI. Phage vector M13mp8 J. Messing:Methods in Enzymology, 101, 20-78 (1983)! replicative form (RF) DNA wasdigested with the restriction enzymes EcoRI and BamHI. The DNA fragmentthus obtained was mixed with the human bFGF DNA fragment derived fromthe pTB669 which was previously digested with EcoRI and BamHI. Themixture was then ligated together by T4 DNA ligase. The ligated DNA wastransformed into infectable cells of Escherichia coli JM105 strain, andthe cells were sown on a plate containing Xga1 as the indicator speciesJ. Messing et al.: Nucleic Acids Research, 9, 309-321 (1981)!. Theplaque containing the recombinant phage (white plaque) was picked up,and the base sequence of the recombinated segment was determined by thedideoxynucleotide synthetic chain termination method J. Messing et al.:Nucleic Acids Research, 9, 309 (1981)!, whereby it was confirmed thathuman bFGF DNA was accurately inserted.

From this M13PO clone was purified single-stranded phage DNA, which wasused as a template for site-directed mutagenesis using syntheticoligonucleotide.

(2) Site-specific mutagenesis

Forty picomoles of the synthetic oligonucleotide: ##STR7## the primerfor converting Cys 26 to Ser (the recognition sequence for restrictionenzyme Rsa I disappears)! was treated with 9 units of T4 kinase at 37°C. for 1 hour in 50 μl of a solution containing 0.1 mM adenosinetriphosphate (ATP), 50 mM hydroxymethylaminomethane hydrochloride(Tris-HCl), pH 8.0, 10 mM MgCl₂ and 5 mM dithiothreitol (DTT). Thiskinase-treated primer (12 picomoles) was heated at 67° C. for 5 minutesand then at 42° C. for 25 minutes in 50 μl of a mixture containing 50 mMNaCl, 1.0 mM Tris-HCl, pH 8.0, 10 mM MgCl₂ and 10 mM β-mercaptoethanol,whereby the primer was hybridized to 5 μg of single-stranded (ss) M13-PODNA. After annealing, the mixture was cooled on ice, and was added to 50μl of a reaction mixture containing 0.5 mM dideoxynucleotidetriphosphate (dNTP), 80 mM Tris-HCl, pH 7.4, 8 mM MgCl₂, 100 mM NaCl, 9units of DNA polymerase I Klenow fragment, 0.5 mM ATP and 2 units of T4DNA ligase, and reaction was carried out at 37° C. for 3 hours and 25°C. for 2 hours. The reaction was terminated by adding 2 μl of 0.2 mMEDTA. The reaction product was used to transform infectable JM 105cells, and the cells were grown overnight. Thereafter, ssDNA wasisolated from the medium's supernatant. Using this ssDNA as the templatefor the second cycle of primer elongation, gel-purified RF DNA wastransformed into infectable JM 105 cells. The cells were sown over anagar plate and cultivated overnight, whereby a phage plaque wasobtained.

(3) Site-directed mutagenesis

The procedure of the above term (2) was repeated, but the syntheticoligonucleotide primer used was ##STR8## which converts cysteine 70 toserine (A recognition sequence for restriction enzyme HaeII isproduced).

(4) Site-directed mutagenesis

The procedure of the above term (2) was repeated, but the syntheticoligonucleotide primer used was ##STR9## which converts cysteine 88 toserine (A recognition sequence for restriction enzyme AluI is produced).

(5) Site-directed mutagenesis

The procedure of the above term (2) was repeated, but theoligonucleotide primer used was ##STR10## which converts cysteine 93 toserine (A recognition sequence for restriction enzyme HinfI isproduced).

(6) Screening and identification of mutated plaques

Plates containing mutated M13-PO plaques above term (1)! and two platescontaining unmutated M13-PO phage plaques were cooled to 4° C., and theplaques from each plate were transferred to two round nitrocellulosefilters by superposing a dry filter on the agar plate for 5 minutes inthe case of the first filter, or by superposing a dry filter for 15minutes in the case of the second filter. Then, the filters were placedon a thick filter paper and immersed in a solution containing 0.2N NaOH,1.5M NaCl and 0.2% Triton X-100, and then on a filter paper in asolution containing 0.5M Tris-HCl, pH 7.5 and 1.5M NaCl for 5 moreminutes, to thereby neutralize the filters. The filters were washed on afilter by immersing them twice in 2×SSC (standard sodium citrate). Thefilters were then allowed to dry in air, after which they were dried at80° C. in a vacuum oven for 2 hours. The duplicated filters weresubjected to prehybridization at 55° C. for 4 hours in 10 ml/filter DNAhybridization buffer (5×SSC), pH 7.0/4×Denhardt's solution(polyvinylpyrrolidone, Ficoll, and bovine serum albumin, 1×=0.02% foreach)/0.1% sodium dodecylsulfate (SDS)/50 mM sodium phosphate buffer, pH7.0/100 μg/ml denatured salmon sperm DNA. The oligonucleotide primer washybridized to 10⁵ cpm/ml at 42° C. for 24 hours. The filters were washedin washing buffers containing 0.1% SDS and decreasing amounts of SSC at50° C. for 30 minutes for each wash. That is, the filters were washedfirst with the buffer containing 2×SSC, and the control filterscontaining unmutated M13-PO plaques were examined for radioactivity bymeans of a Geiger counter. While reducing SSC concentration step bystep, the filters were washed until no detectable radioactivity remainedon the control filters containing unmutated M13-PO plaques. The minimumSSC concentration used was 0.1×SSC. The filters were air-dried andexposed to film at -70° C. for 2 to 3 days to thereby takeradioautograms. A total of 10,000 mutated M13-PO plaques and 100unmutated control plaques were screened by means of the kinase-treatedoligonucleotide probe. None of the control plaques hybridized to theprobe, while 3 to 10 of the mutated M13-PO plaques hybridized to theprobe.

One of the mutated M13-O plaques was picked up and inoculated to JM105medium. From the supernatant was prepared ssDNA, and from the cellpellet was prepared double-stranded (ds) DNA. Using appropriateoligonucleotide primers and ssDNAs, the base sequences were analyzed.

As a result, it was respectively confirmed that TGC (Cys26) codon hadbeen converted to TCT (Ser) codon, TGT (Cys7O) codon had been convertedto AGC (Ser) codon, TGT (Cys88) codon had been converted to TCT (Ser)codon, and TGT (Cys93) codon had been converted to TCT (Ser) codon.

Of the mutated M13-PO phages, the phage in which the codon Cys-26 hadbeen converted to Ser was designated M13-P1; the phage in which thecodon Cys-70 had been Ser, M13-P2; the phage in which the codon Cys-88had been converted to Ser, M13-P3; and the phage in which the codonCys-93 had been converted to Ser, M13-P4.

Reference Example 5

(Expression of human bFGF mutein-encoding gene in Escherichia coli )

(1) Construction of human bFGF mutein expression plasmid pTB739)

The M13-P1 replicative form (RF) obtained above in Reference Example 4was cleaved with the restriction enzymes EcoRI and PstI to therebyobtain an about 0.5 kb DNA fragment including the human bFGFmutein-encoding region.

Separately, the trp promoter-containing plasmid ptrp781 Kurokawa, T. etal.: Nucleic Acids Res., 11, 3077-3085 (1983)! was cleaved withEcoRI-PstI, and an about 3.2 kb DNA fragment containing the trppromoter, the tetracycline resistance gene and the plasmid replicationorigin was isolated. This 3.2 kb DNA fragment was ligated with theabove-mentioned 0.5 kb EcoRI-PstI DNA fragment containing the human bFGFmutein-encoding gene region by T4 DNA ligase reaction, whereby a humanbFGF mutein expression plasmid, pTB739, was constructed.

This plasmid pTB739 was used to transform Escherichia coli DH1 to give atransformant carrying the plasmid pTB739 containing the mutein-encodinggene, namely Escherichia coli DH1/pTB739 (IFO 14575, FERM BP-1641).

(2) Preparation of cell extract

The above transformant was cultivated in M9 medium containing 1%glucose, 0.4% casamino acid and 8 μg/ml tetracycline. When the Klettvalue was about 200, 3-β-indolylacrylic acid was added to 25 μg/ml, andthe cultivation was continued for 4 more hours. Thereafter, cells wereharvested and suspended in one twentieth volume of 10% sucrose solutionin 20 mM Tris-HCl, pH 7.6. To this suspension were addedphenylmethylsulfonyl fluoride (PMSF) to 1 mM (final concentration), EDTAto 10 mM, NaCl to 0.1M, spermidine hydrochloride to 10 mM and lysozymeto 100 μg/ml. After allowing to stand at 0° C. for 45 minutes, the wholemixture was sonicated for 30 seconds. The sonication product wascentrifuged at 18,000 rpm (Sorvall centrifuge, SS 34 rotor, U.S.A.) for30 minutes to give a supernatant, which was used as the cell extract.

(3) Human bFGF activity of cell extract

A Nunc 96-well microtiter plate (flat bottomed) was sown with mouseBALB/c3T3 cells (2×10³ cells per well) with DMEM medium containing 5%calf serum (0.2 ml per well) and the cells were cultured. Next day, themedium was replaced with DMEM medium containing 0.5% calf serum. After 3days of cultivation, dilutions of the cell extract as prepared by serial5-fold dilution with DME medium containing 0.5% BSA were added in anamount of 10 μl per well. After 20 hours of continued cultivation, 2 μlof ³ H-Tdr (5 Ci/mmol, 0.5 mCi/ml RCC Amersham) was added to each well.Six hours later, cells in each well were scraped off by treatment withphosphate buffer (PBS) containing 0.2% trypsin and 0.02% EDTA andcollected on a glass filter using a Titertek cell harvester, and thequantity of ³ H-Tdr taken up by the cells was measured using ascintillation counter.

The cell extract of E. coli DH1/pTB739 thereby tested showed FGFactivity.

The mutein CS1 in which the 26-position Cys of human bFGF had beenreplaced by Ser was thus obtained.

Reference Example 6

(Expression in Escherichia coli of gene encoding human bFGF mutein)

(1) Construction of the plasmid pTB742 for human bFGF mutein expression

The M13-P2 replicative form (RF) obtained in Reference Example 4 abovewas cleaved using the restriction enzymes EcoRI and PstI to obtain anabout 0.5 kb DNA fragment containing a region which encodes a human bFGFmutein.

Separately, a plasmid ptrp781 DNA containing a trp promoter was cleavedusing EcoRI-PstI to separate an about 3.2 kb DNA fragment containing atrp promoter, a tetracycline resistance gene and a plasmid replicationinitiation site. This 3.2 kb DNA fragment and the above-mentioned 0.5 kbEcoRI-PstI DNA fragment containing a gene region encoding a human bFGFmutein were ligated together by T4 DNA ligase reaction to construct theplasmid pTB742 for the expression of a human bFGF mutein.

Using this plasmid pTB742, Escherichia coli DH1 was transformed, wherebythe strain Escherichia coli DH1/pTB742 (IFO 14584, FERM BP-1642) wasobtained, which harbors the plasmid pTB742 containing themutein-encoding gene.

(2) Preparation of bacterial cell extract

The above-mentioned transformant was cultured by the method described inReference Example 5 (2) to give a supernatant, which was then used as abacterial cell extract.

(3) Human bFGF activity of the bacterial cell extract

A determination was made of the human bFGF activity on the bacterialcell extract obtained in (2) above, by the method described in ReferenceExample 5 (3).

The bacterial cell extract of E. coli DH1/pTB742 thereby testedexhibited FGF activity.

The mutein CS2, in which Cys at the 70-position of human bFGF had beenreplaced by Ser, was thus obtained.

Reference Example 7

(Expression in Escherichia coli of gene encoding human bFGF mutein)

(1) Construction of the plasmid pTB743 for human bFGF mutein expression

The M13-P3 replicative form (RF) obtained in Reference Example 4 abovewas cleaved using the restriction enzymes EcoRI and PstI to obtain anabout 0.5 kb DNA fragment containing a region which encodes a human bFGFmutein.

Separately, a plasmid ptrp781 DNA containing a trp promoter was cleavedusing EcoRI-PstI to separate an about 3.2 kb DNA fragment containing atrp promoter, a tetracycline resistance gene and a plasmid replicationinitiation site. This 3.2 kb DNA fragment and the above-mentioned 0.5 kbEcoRI-PstI DNA fragment containing a gene region encoding a human bFGFmutein were ligated together by T4 DNA ligase reaction to construct theplasmid pTB743 for the expression of human bFGF mutein.

Using this plasmid pTB743, Escherichia coli DH1 was transformed, wherebythe strain Escherichia coli DH1/pTB743 (IFO 14585, FERM BP-1643) wasobtained, which harbors the plasmid pTB743 containing themutein-encoding gene.

(2) Preparation of bacterial cell extract

The above-mentioned transformant was cultured in the manner described inReference Example 5 (2) to give a supernatant, which was then used as abacterial cell extract.

(3) Human bFGF activity of the bacterial cell extract

A determination was made of the human bFGF activity of the bacterialcell extract obtained in (2) above, by the method described in ReferenceExample 5 (3).

The bacterial cell extract of E. coli DH1/pTB743 thereby testedexhibited FGF activity.

The mutein CS3, in which Cys at the 88-position of human bFGF had beenreplaced by Ser, was thus obtained.

Reference Example 8

(Expression in Escherichia coli of gene which encodes human bFGF mutein)

(1) Construction of the plasmid pTB744 for human bFGF mutein expression

The M13-P4 replicative form (RF) obtained in Reference Example 4 abovewas cleaved using the restriction enzymes EcoRI and PstI to obtain anabout 0.5 kb DNA fragment containing a region which encodes a human bFGFmutein.

Separately, a plasmid ptrp781 DNA containing a trp promoter was cleavedusing EcoRI-PstI to separate an about 3.2 kb DNA fragment containing atrp promoter, a tetracycline resistance gene and a plasmid replicationinitiation site. This 3.2 kb DNA fragment and the above-mentioned 0.5 kbEcoRI-PstI DNA fragment containing a gene region encoding a human bFGFmutein were ligated together by T4 DNA ligase reaction to construct theplasmid pTB744 for the expression of a human bFGF mutein.

Using this plasmid pTB744, Escherichia coli DH1 was transformed, wherebythe strain Escherichia coli DH1/pTB744 (IFO 14586, FERM BP-1644) wasobtained, which harbors the plasmid pTB744 containing themutein-encoding gene.

(2) Preparation of bacterial cell extract

The above-mentioned transformant was cultured by the method described inReference Example 5 (2) to give a supernatant, which was then used as abacterial cell extract.

(3) Human bFGF activity of the bacterial cell extract

A determination was made of the human bFGF activity of the bacterialcell extract obtained in (2) above, by the method described in ReferenceExample 5 (3).

The bacterial cell extract from E. coli DH1/pTB744 thereby testedexhibited FGF activity.

The mutein CS4, in which Cys at the 93-position in human bFGF had beenreplaced by Ser was thus obtained.

Reference Example 9

(Screening and identification of plaques which were made mutagenic)

Plates containing mutated M13-P2 phage plaques obtained in ReferenceExample 4 and two plates containing unmutated M13-P2 phage plaquesobtained in Reference Example 4 were cooled to 4° C., and the plaquefrom each plate was transferred to 2 round nitrocellulose filters bykeeping a dry filter placed on the agar plate for 5 minutes in the caseof the 1st filter, and for 15 minutes in the case of the 2nd filter. Thefilters were then kept placed for 5 minutes on thick filter papersimmersed in 0.2N NaOH, 1.5M NaCl and 0.2% Triton X-100, after which theywere neutralized by keeping them placed for 5 more minutes on filterpapers immersed in 0.5M Tris-HCl (pH 7.5) and 1.5M NaCl. The filterswere twice washed on filters immersed in 2×SSC (standard sodium citrate)in the same manner, and were allowed to dry, and this was followed bydrying at 80° C. for 2 hours in a vacuum oven. The overlapped filterswere subjected to prehybridization at 55° C. for 4 hours with 10ml/filter of a DNA hybridization buffer solution (5×SSC) having apH-value of 7.0 containing 4×Denhardt's solution (polyvinylpyrrolidone,Ficoll and bovine serum albumin, 1×=0.02%), 0.1% sodium dodecyl sulfate(SDS), 50 mM sodium phosphate-buffered solution having a pH-value of 7.0and 100 μg/ml denatured salmon sperm DNA. Hybridization was carried outat 42° C. for 24 hours with 10⁵ cpm/ml of an oligonucleotide primer. Thefilters were each washed in a buffer solution for washing containing0.1% SDS and a reduced amount of SSC at 50° C. for 30 minutes. Thefilters were then first washed with a buffer solution containing 2×SSC;the control filters, which contained unmutated M13-P2 plaques, wereexamined for radioactivity using a Geiger counter. While stepwisereducing SSC concentration, the control filters were washed until nodetectable radioactivity remained on the filters. The minimum of theused SSC concentrations was 0.1×SSC. The filters were allowed to dry inair, and radioautographs were taken by 2 to 3 days of exposure at -70°C. Screening was carried out of 10,000 mutated M13-P2 plaques and 100unmutated control plaques using a kinase-treated oligonucleotide probe.None of the control plaques hybridized to the probe, while 3 to 10 ofthe mutated M13-P2 plaques hybridized to the probe.

One of the mutated M13-P2 plaques was taken, and was inoculated to aJM105 culture medium. From the resulting supernatant an ssDNA wasprepared, and from the bacterial cell pellets a double-stranded (ds) DNAwas prepared. Analyses were made of the base sequences using appropriateoligonucleotide primers and ssDNAs.

As a result, it was respectively confirmed that the TGC (Cys-26) codonhad been changed to a TCT (Ser) codon; the TGT (Cys-88) codon, to a TCT(Ser) codon; and the TGT (Cys-93) codon, to a TCT (Ser) codon.

Of the mutated M13-P2 phages, the phage in which the codons Cys-26 and-70° had become Ser-encoding codons was named M13-P12; the phage inwhich the codons Cys-70 and -88 had become Ser-encoding codons, M13-P23;and the phage in which the codons Cys-70 and -93 had become Ser-encodingcodons, M13-P24.

Reference Example 10

(Expression in Escherichia coli of gene encoding human bFGF mutein)

(1) Construction of the plasmid pTB762 for human bFGF mutein expression

The M13-P23 replicative form (RF) obtained in Reference Example 9 abovewas treated in the manner described in Reference Example 5 (1) toconstruct the plasmid pTB762 for human bFGF mutein expression.

Using this plasmid pTB762, Escherichia coli MM294 was transformed,whereby the strain Escherichia coli MM294/pTB762 (IFO 14613, FERMBP-1645) was obtained, which harbors the plasmid pTB762 containing themutein-encoding gene.

(2) Preparation of bacterial cell extract

The above-mentioned transformant was cultured by the method described inReference Example 5 (2) to give a supernatant, which was then used as abacterial cell extract.

(3) Human bFGF activity of the bacterial cell extract

A determination was made of the human bFGF activity of the bacterialcell extract obtained in (2) above, by the method described in ReferenceExample 5 (3).

The bacterial cell extract from E. coli MM294/pTB762 thereby testedexhibited FGF activity.

The mutein CS23, in which Cys at the 70-position and at the 88-positionhad been replaced by Ser, was thus obtained.

Reference Example 11

(Production of recombinant DNAs having mutein-encoding base sequence)

(1) Cloning of M13 vector for human bFGF gene

The plasmid pTB669 obtained in Reference example 2 was digested with therestriction enzymes EcoRI and BamHI. Phage vector M13mp8 J. Messing,Methods in Enzymology, 101, 20˜78 (1983)! replicative form (RF) DNA wasmixed with a human bFGF DNA fragment derived from pTB669, previouslydigested with EcoRI and BamHI. The mixture was then subjected toligation using T4 DNA ligase. The resulting ligated DNA was transformedinto infectable cells of the strain Escherichia coli JM105; thetransformant cells were spread over a plate whose indicator species wasXgal J. Messing et al., Nucleic Acids Research, 9, 309-321 (1981)!; theplaque containing the recombinant phage (white plaque) was picked up;the base sequence of the recombinated region was determined by thedideoxynucleotide synthesis chain termination method J. Messing et al.,Nucleic Acids Research, 9, 309 (1981)!, whereby it was confirmed thatthe human bFGF DNA had been accurately inserted.

From this M13-PO clone was purified a single-stranded phage DNA, whichwas used as a template for site-directed mutagenesis using a syntheticoligonucleotide.

(2) Site-specific mutagenesis

In the presence of 0.1 mM adenosine triphosphate (ATP), 50 mMhydroxymethylaminomethane hydrochloride (Tris-HCl) having a pH-value of8.0, 10 mM MgCl₂, 5 mM dithiothreitol (DTT) and 9 units of T4 kinase, ina total amount of 50 μl, 40 picomoles of the synthetic oligonucleotide:

5'-CGGGCATGAATTCGCCGCT-3'

primer for producing in the base sequence a recognition site for therestriction enzyme EcoRI, and subsequently changing Pro-14 to Met! wastreated with T4 kinase at 37° C. for 1 hour. This kinase-treated primer(12 picomoles) was heated at 67° C. for 5 minutes, and at 42° C. for 25minutes, in 50 μl of a mixture containing 50 mM NaCl, 1.0 mM Tris-HClhaving a pH-value of 8.0, 10 mM MgCl₂ and 10 mM β-mercaptoethanol,whereby it was hybridized to 5 μg of the single-stranded (ss) M13-PODNA. The annealed mixture was then cooled on ice, and was added to 50 μlof a reaction mixture containing 0.5 mM deoxynucleotide triphosphate(dNTP), 80 mM Tris-HCl having a pH-value of 7.4, 8 mM MgCl₂, 100 mMNaCl, 9 units of DNA polymerase I Klenow fragment, 0.5 mM ATP and 2units of T4 DNA ligase, and reaction was carried out at 37° C. for 3hours, and at 25° C. for 2 hours, whereafter the reaction was stopped byadding 2 μl of 0.2 mM EDTA. The reaction product was used to transforminfectable JM 105 cells; the transformant cells were allowed to growovernight, whereafter an ssDNA was isolated from the culture mediumsupernatant. Using this ssDNA as a template for the 2nd cycle of primerelongation, gel-purified RF-type DNA was transformed into infectable JM105 cells; the resulting transformant cells were spread over an agarplate, and were cultured overnight to obtain phage plaques.

(3) Site-directed mutagenesis

The procedure of the above term (2) was repeated but the syntheticoligonucleotide primer used was: 5'-CGCCCATGGTGCCATCCTC-3' whichproduces in the base sequence a recognition site for the restrictionenzyme NcoI, and concurrently changes Gly-9 to Thr and Ser-10 to Met,respectively:

(4) Site-directed mutagenesis

The procedure of the above term (2) was repeated but the syntheticoligonucleotide primer used was: 5'-TAACACCTTAAGAAGCCAG-3' whichproduces in the base sequence a recognition site for the restrictionenzyme Af1II, and concurrently changes the Lys-87-encoding codon to atermination codon.

(5) Site-directed mutagenesis

The procedure of the above term (2) was repeated but the syntheticoligonucleotide primer used was: 5'-CCGGACTCCGTTAACTCGG-3' whichproduces in the base sequence a recognition site for the restrictionenzyme HpaI, and concurrently changes Asp-42 to Asn.

(6) Site-directed mutagenesis

The procedure of the above term (2) was repeated but the syntheticoligonucleotide primer used was: 5'-CTTCTCCTGGACTCCGTCAAC-3' whichdeletes the recognition site for the restriction enzyme HpaII in thebase sequence, and concurrently changes Arg-45 to Gln.

(7) Screening and identification of plaques which were mutagenic

Plates containing mutated M13-PO plaques above term (1)! and 2 platescontaining unmutated M13-PO phage plaques were cooled to 4° C., and theplaques from each plate were transferred to 2 round nitrocellulosefilters by keeping a dry filter placed on the agar plate for 5 minutesin the case of the 1st filter, and for 15 minutes in the case of the 2ndfilter. The filters were then kept placed for 5 minutes on thick filterpapers immersed in 0.2N NaOH, 1.5M NaCl and 0.2% Triton X-100, afterwhich they were neutralized by keeping them placed for 5 more minutes onfilter papers immersed in 0.5M Tris-HCl having a pH-value of 7.5 and1.5M NaCl. The filters were twice washed on filters immersed in 2×SSC(Standard Sodium Citrate) in the same manner, and were allowed to dry,and this was followed by drying at 80° C. for 2 hours in a vacuum oven.The overlapped filters were subjected to prehybridization at 55° C. for4 hours with 10 ml/filter of a DNA hybridization buffer solution (5×SSC)having a pH-value of 7.0 containing 4×Denhardt's solution(polyvinylpyrrolidone, Ficoll and bovine serum albumin, 1×=0.02%), 0.1%sodium dodecyl sulfate (SDS), 50 mM sodium phosphate-buffered solutionhaving a pH-value of 7.0 and 100 μg/ml denatured salmon sperm DNA.Hybridization was carried out at 42° C. for 24 hours with 10⁵ cpm/ml ofan oligonucleotide primer. Each filter was washed at 50° C. for 30minutes in a buffer solution for washing containing 0.1% SDS and areduced amount of SSC. The filters were then first washed with a buffersolution containing 2×SSC; the control filters, which containedunmutated M13-PO plaques, were examined for radioactivity using a Geigercounter. While stepwise reducing SSC concentration, the control filters,which contained unmutated M13-PO plaques, were washed until nodetectable radioactivity remained on the filters. The minimum of theused SSC concentrations was 0.1×SSC. The filters were allowed to dry inair, and autoradiographs were taken by 2 to 3 days of exposure at -70°C. Screening was carried out of 10,000 mutated M13-PO plaques and 100unmutated control plaques by means of an oligonucleotide probe treatedwith ³² P-γ-ATP. None of the control plaques hybridized to the probe,while 3 to 10 of the mutated M13-PO plaques hybridized to the probe.

One of the mutated M13-PO plaques was taken, and was inoculated to aJM105 culture medium. From the resulting supernatant an ssDNA wasprepared, and from the bacterial cell pellets a double-stranded (ds) DNAwas prepared. Analyses were made of the base sequences using appropriateoligonucleotide primers and ssDNAs.

As a result, it was respectively confirmed that the GGC (Gly-9) codonhad been changed to an ACC (Thr) codon and the AGC (Ser-10) codon hadbeen changed to an ATG (Met) codon; the CCG (Pro-14) codon, to an ATG(Met) codon; the AAA (Lys-87) codon, to a TAA (termination) codon; theGAC (Asp-42) codon, to an AAC (Asn-42) codon; and the CGG (Arg-45)codon, to a CAG (Gln-45) codon.

Of the mutated M13-PO phages, the phage in which Gly-9 codon had becomea Thr-encoding codon and the Ser-10 codon had become a Met-encodingcodon was named M13-PN10;

the phage in which the Pro-14 codon had become a Met-encoding codon,M13-PN14;

the phage in which the Lys-87 codon had become a termination codon,M13-PC86;

the phage in which the Asp-42 codon had become an Asn-encoding codon,M13-PDN42; and,

the phage in which the Arg-45 codon had become a Gln-encoding codon,M13-PRQ45.

Reference Example 12

(Expression in Escherichia coli of gene which encodes human bFGF mutein)

(1) Construction of the plasmid pTB795 for human bFGF mutein expression

The M13-PN14 replicative form (RF) obtained in Example 11 above wastreated in the manner described in Reference Example 5 (1) to constructthe plasmid pTB795 for human bFGF mutein.

Using this plasmid pTB795, Escherichia coli MM294 was transformed,whereby the strain Escherichia coli MM294/pTB795 (IFO 14700, FERMBP-1660) was obtained, which contains the plasmid pTB795 containing themutein-encoding gene.

(2) Preparation of bacterial cell extract

The above-mentioned transformant was cultured by the method described inReference Example 5 (2) to give a supernatant, which was then used as abacterial cell extract.

(3) Human bFGF activity of the bacterial cell extract

A determination was made of the human bFGF activity of the bacterialcell extract obtained in (2) above, by the method described in ReferenceExample 5 (3).

The bacterial cell extract from E. coli MM294/pTB795 thereby testedexhibited FGF activity. The mutein N14, in which the amino acid sequenceof from Pro at the 2-position to Pro at the 14-position of human bFGFhad been deleted, was thus obtained.

Reference Example 13

(Expression in Escherichia coli of gene which encodes human bFGF mutein)

(1) Construction of the plasmid pTB796 for human bFGF mutein expression

The M13-PC86 replicative form (RF) obtained in Reference Example 11above was treated in the manner described in Reference Example 5 (1) toconstruct the plasmid pTB796 for human bFGF mutein.

Using this plasmid pTB796, Escherichia coli MM294 was transformed,whereby the strain Escherichia coli MM294/pTB796 (IFO 14701, FERMBP-1661) was obtained, which contains the plasmid pTB796 containing themutein-encoding gene.

(2) Preparation of bacterial cell extract

The above-mentioned transformant was cultured by the method described inReference Example 5 (2) to give a supernatant which contains the muteinC86, in which the amino acid sequence of from Lys at the 87-position toSer at the 147-position had been deleted, and the supernatant was thenused as a bacterial cell extract.

Reference Example 14

(Expression in Escherichia coli of gene which encodes human bFGF mutein)

(1) Construction of the plasmid pTB797 for human bFGF mutein expression

The M13-PDN42 replicative form (RF) obtained in Reference Example 11above was treated in the manner described in Reference Example 5 (1) toconstruct the plasmid pTB797 for human bFGF mutein.

Using this plasmid pTB797, Escherichia coli MM294 was transformed,whereby the strain Escherichia coli MM294/pTB797 (IFO 14702, FERMBP-1662) was obtained, which harbors the plasmid pTB797 containing themutein-encoding gene.

(2) Preparation of bacterial cell extract

The above-mentioned transformant was cultured by the method described inReference Example 5 (2) to give a supernatant, which was then used as abacterial cell extract.

(3) Human bFGF activity of the bacterial cell extract

A determination was made of the human bFGF activity of the bacterialcell extract obtained in (2) above, by the method described in ReferenceExample 5 (3).

The bacterial cell extract from E. coli MM294/pTB797 thereby testedexhibited FGF activity. The mutein DN42, in which Asp at the 42-positionof human bFGF had been replaced by Asn, was thus obtained.

Reference Example 15

(Expression in Escherichia coli of gene which encodes human bFGF mutein)

(1) Construction of the plasmid pTB855 for human bFGF mutein expression

The DNA of the plasmid pTB669 which was obtained in the above mentionedReference Example 2 was cleaved with a restriction enzyme HincII, and itwas ligated with EcoRI linker p(5'CATGAATTCATG 3') under T4 DNA ligasereaction. Thus obtained DNA was further cleaved with a restrictionenzymes EcoRI and PstI to recover a DNA fragment of about 0.35 kb. ThisDNA fragment was ligated with the about 3.2 kb DNA fragment obtained inReference Example 5 (1), the fragment being obtained by cleaving theplasmid ptrp781 with EcoRI-PstI, to obtain the plasmid pTB855 for humanbFGF mutein expression was constructed.

Using this plasmid 855, Escherichia coli MM294 was transformed, wherebythe strain Escherichia coli MM294/pTB855, which contains the plasmidpTB855 having the mutein-encoding gene.

(2) Preparation of bacterial cell extract

The above-mentioned transformant was cultured by the method described inReference Example 5 (2) to give a supernatant, which was then used as abacterial cell extract.

(3) Human bFGF activity of the bacterial cell extract

A determination was made of the human bFGF activity of the bacterialcell extract obtained in (2) above, by the method described in ReferenceExample 5 (3).

The bacterial cell extract from E. coli MM294/pTB855 thereby testedexhibited FGF activity. The mutein N41, in which the amino acid sequenceof from Pro at the 2-position to Val at the 41-position of human bFGFhad been deleted, was thus obtained.

Reference Example 16

(Expression in Escherichia coli of gene which encodes human bFGF mutein)

(1) Construction of the plasmid pTB856 for human bFGF mutein expression

The DNA of the plasmid pTB669 which was obtained in the above mentionedReference Example 2 was partly cleaved with a restriction enzyme BamHIso as to obtain BamHI recognition site in the bFGF gene. The site wasfurther cleaved with Escherichia coli DNA polymerase I in the presenceof dATP, dCTP, dGTP, dTTP to give blunt end. This DNA is ligated withNheI linker p(5'CTAGCTAGCTAG3') under T4 DNA ligase reaction. Aftertreating with the restriction enzyme NheI and ligating the cleaved siteunder T4 DNA ligase reaction, the plasmid pTB856 for human bFGF muteinexpression was constructed.

Using this plasmid pTB856, Escherichia coli MM294 was transformed,whereby the strain Escherichia coli MM294/pTB856 which contains theplasmid pTB856 having the mutein-encoding gene.

(2) Preparation of bacterial cell extract

The above-mentioned transformant was cultured by the method as inReference Example 5 (2) to give a supernatant which contains the muteinC129, in which the amino acid sequence of from Lys at the 130-positionto Ser at the 147-position had been deleted, was thus obtained, and thenthe supernatant which was then used as a bacterial cell extract.

Reference Example 17

(Production of H-Leu-Pro-Met-Ser-Ala-Lys-Ser-OH)

Boc-Ser(Bzl)-resin (696 mg, 0.72 m mol/g resin) was applied to automaticpeptide synthesizer Type 430A (Applied Biosystems, U.S.A.), and thefollowing amino acids were applied to the synthesizer in that order soas to cause condensation reaction:

Boc-Lys(Z)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH, Boc-Met-OH, Boc-Pro-OII,

Boc-Leu-OH

Bzl: benzyl

Boc: t-butoxycarbonyl

Z: benzyloxycarbonyl

By said procedure, 1.08 g ofBoc-Leu-Pro-Met-Ser(Bzl)-Ala-Lys(Z)-Ser(Bzl)-resin was produced. 400 mgof the peptide-resin was incubated in 5.0 ml of hydrogen fluoridecontaining 0.5 ml of anisole and 0.5 ml of dimethylsulfide at 0° C. for60 minutes to recover the peptide from resin. The excess amount ofhydrogen fluoride was removed by distillation under reduced pressure togive a residue. The residue was washed with diethyl ether, and extractedwith 30 ml of water, and lyophilized. The lyophylizate was dissolved in5 ml of water, and the solution was subjected to ion-exchange employingAmberlite IRA-400 (acetate form) resin (column 2×5 cm, elution solvent:water). The eluate was concentrated under reduced pressure, andsubjected to gel filtration with Sephadex LH-20 (Pharmacia, column2.5×125 cm, elution solvent: 1N acetic acid) to obtain the peptideH-Leu-Pro-Met-Ser-Ala-Lys-Ser-OH.

Yield: 118 mg (78.8%)

Rf value: 0.22 (ethyl acetate:acetic acid:butanol:water=1:1:1:1)

α!_(D) ²⁵ -8.1 (c=0.11, 1N acetic acid)

Amino acid analysis: Ser. 2.08, Pro 1.06, Ala 1.00, Met 0.98, Leu 1.03,Lys 0.99.

Example 1

(Immunization)

BALB/c mice (female, 4-week old) had the antigen human bFGF (as obtainedin Reference Example 3) in solution in 0.4 ml of Freund's completeadjuvant (Difco Laboratories, U.S.A.) injected intraperitoneally. Threeweeks later, 10 μg of the antigen hbFGF in solution in 0.4 ml ofFreund's incomplete adjuvant was intraperitoneally administered. 3 weekslater, the same additional immunization was carried out, and two weekslater, 10 μg of human bFGF in saline was intraperitoneally inoculated.

Example 2

(1) Cell fusion

From the immunized mice mentioned in Example 1, the spleen was excised 4days after final antigen challenge to thereby obtain cells to be usedfor cell fusion. These cells were suspended in a medium prepared bymixing together Isokov medium and Ham F-12 medium in a ratio of 1:1(hereinafter referred to as IH medium).

The mouse myeloma cell P3-X63-Ag 8UI was subcultured in RPMI 1640 mediumcontaining 10% fetal bovine serum under an atmosphere of 5% carbondioxide and 95% air.

Cell fusion was conducted in accordance with the method established byKohler and Milstein Kohler, G. and Milstein, C.: Nature, 256, 495(1975)!. 2.9×10⁷ cells of the above myeloma cell line and 1.5×10⁸immunized lymphocytes obtained by the above-mentioned method were mixedtogether and centrifuged, and 45% polyethylene glycol 6000 (hereinafterreferred to as PEG 6000) in 0.3 ml of IH medium was dropwise added. ThePEG 6000 solution was preheated to 37° C., and was gradually added. Fiveminutes later, the 37° C.-preheated IH medium was added at a rate of 0.5ml per minute to make 10 ml. The solution was then centrifuged at roomtemperature at 600 rpm for 15 minutes, and the supernatant was removed.This cell precipitate was suspended in 200 ml of IH medium containing20% calf serum, and this suspension was transferred to a 24-wellmicroplate (Linbro) in an amount of 2 ml per well. One day later, IHmedium (containing 20% calf serum) supplemented with HAT (1×10⁻⁴Mhipoxanthine, 4×10⁻⁷ M aminopterin, 1.6×10⁻⁵ M thymidine) (hereinafterreferred to as HAT medium) was added to the microplate in an amount of 1ml per well, and, a further three day, one half amount of the medium wasreplaced with HAT medium. The cells thus grown are hybrid cells.

(2) Search for antibody-producing cells

Previously, the hybridoma conditioned medium was added in an amount of100 μl per well to a 96-well polystyrene microtiter plate which had hadhuman bFGF immobilized thereto, and incubation was carried out at roomtemperature for 2 hours. The medium was removed, and, after washing, thehorse radish peroxidase (HRP)-labeled anti-mouse IgG goat antibody(Miles) was added as the secondary antibody, and incubated at roomtemperature for 2 hours. The secondary antibody was removed, and, afterthoroughly washing the wells, coloring reaction was carried out in thepresence of added reaction substrate (EIA method). By this method potentvalency was observed in 3 wells.

(3) Cloning of hybrid cells

Cells in each of these three cells were sown to 0.5 cell per well to a96-well microtiter plate which had had 10⁴ cells/well mouse thymocytesas vegetative cells sown thereon, and cloning was carried out. As aresult, three clones, namely the mouse HbF99 cell (IFO 50122), the mouseHbF161 cell (IFO 50123) and the mouse HbF165 cell (IFO 50124) wereobtained.

The results of the determination of antibody titers in supernatants ofthese cell lines are shown in Table 2.

                  TABLE 2    ______________________________________    Culture Supernatant     bFGF-           HbF    HbF    HbF  Parent Line                                        Immunized Mouse    Dilution           99     161    165  Myeloma Cell                                        Serum    ______________________________________     ×64           1.93   1.08   0.66 0.02      --     ×128           1.72   0.63   0.51 0.02      --    ×3200           --     --     --   --        1.93    ×6400           --     --     --   --        1.25    ______________________________________     Note: The numerical figures in the above Table 2 represent absorbances at     492 nm wavelength; blank (--) means that determination was not made.

The cloned cells were stored in IH medium containing 20% calf serum andhaving dimethylsulfoxide (DMSO) added thereto to 10% under liquidnitrogen atmosphere.

Example 3

(Immunoglobulin class of monoclonal antibodies)

The mouse antibodies obtained in Example 2 were reacted with variousimmunoglobulin standards by means of the Mouse-Typer subisotyping kit(Bio-Rad). The results are presented in Table 3.

                  TABLE 3    ______________________________________    Immunoglobulin               Monoclonal Antibodies According to the Invention    Standard   MoAb99     MoAb161     MoAb165    ______________________________________    IgG 1      -          -           -    IgG 2a     -          -           -    IgG 2b     -          -           -    IgG 3      -          -           -    IgM        +          +           +    IgA        -          -           -    ______________________________________     Note: + indicates positive for the reaction, and - indicates negative for     the reaction.

From Table 2 it is obvious that HbF99, HbF161, and HbF165, all belong tothe immunoglobulin class IgM.

Example 4

Spleens were collected from BALB/c mice immunized by the methoddescribed in Example 1, and, by the methods described in Example 2 (1),(2) and (3), the hybridomas HbF12 (IFO 50142), HbF45, HbF47, HbF52 (IFO50143), HbF78 (IFO 50144) and HbF98 (IFO 50145) were obtained. 2×10⁶cells each hybridoma were intraperitoneally inoculated to mice to which0.5 ml of mineral oil were preinjected.

After 10 days, 2 to 4 ml of ascites per mouse were collected, andmonoclonal antibodies MoAb12, MoAb45, MoAb47, MoAb52, MoAb78 and MoAb98were obtained, from said hybridomas, respectively, in accordance withthe method described in Example 2 (4).

By the method described in Example 3, determinations were made of theimmunoglobulin class of said monoclonal antibodies, whereby thefollowing results shown in Table 4 were obtained.

                  TABLE 4    ______________________________________    Monoclonal Antibody                    Immunoglobulin Class    ______________________________________    MoAb12           IgG 1    MoAb45           IgG 1    MoAb47           IgM    MoAb52           IgG 2b    MoAb78           IgG 2b    MoAb98           IgG 1    ______________________________________

Example 5

(1) Preparation of radiolabeled hbFGF

Using the transformant Escherichia coli MM294/pTB669 (IFO 14532, FERMBP-1281) described in Reference Example 2, hbFGF radiolabeled with ³⁵ Swas obtained in the following manner.

The above Escherichia coli MM294/pTB669 was cultivated in the mediumdescribed in Reference Example 3 until the Klett value was 200. Thisculture broth in the one fifth amount was poured into M9 (Met⁻) medium.The M9 (Met⁻) medium was prepared by supplementing the M9 mediumcontaining 1% glucose, 8 μg/ml tetracycline, and the amino acid shownbelow:

    ______________________________________    Amino acid composition    ______________________________________    L-alanine             25.0     mg/l    L-arginine hydrochloride                          84.0     mg/l    L-asparagine monohydrate                          28.4     mg/l    L-aspartic acid       30.0     mg/l    L-cysteine disodium salt                          82.8     mg/l    L-glutamic acid       75.0     mg/l    L-glutamine           584.0    mg/l    L-glycine             30.0     mg/l    L-histidine hydrochloride monohydrate                          42.0     mg/l    L-isoleucine          105.0    mg/l    L-leucine             105.0    mg/l    L-lysine hydrochloride                          146.0    mg/l    L-phenylalanine       66.0     mg/l    L-proline             40.0     mg/l    L-serine              42.0     mg/l    L-threonine           95.0     mg/l    L-tyrosine            83.9     mg/l    L-valine              94.0     mg/l    ______________________________________

Cultivation was carried out in the M9 (Met⁻) medium until the Klettvalue was 200, and 3-β-indoleacrylic acid was added to 25 μg/ml, and thecultivation was continued for 2 more hours. Thereafter, a 1-ml portionof the culture broth was collected, and 10 μCi of ³⁵ S-Met (specificactivity >1000 Ci/mmol) was added, and cultivation was carried out for30 minutes. After cultivation, cells were harvested, and a cell extractwas obtained in accordance with the method described in ReferenceExample 3.

The Escherichia coli MM294 carrying the vector plasmid ptrp781 anddescribed in Reference Example 2 was subjected to the same procedure tothereby obtain a labeled cell extract.

(2) Imnunoprecipitation

A 10% solution of Protein A (BRL) was prepared in accordance with theinstruction manual thereof. An unlabeled Escherichia coli cell extractwas obtained by treating Escherichia coli MM294/ptrp781 by the method ofReference Example 3.

One milliliter of the ascites fluid obtained in Example 4 was mixed with100 μl of the unlabeled Escherichia coli cell extract, and the mixture,after being allowed to stand at 4° C. for 1 hour, had 10⁶ cpm of thelabeled cell extract (MM294/ptrp781 or MM294/pTB669) added thereto, andwas allowed to stand at 4° C. overnight.

To 100 μl of the 10% solution of Protein A, was added 100 μl of theunlabeled Escherichia coli cell extract, and the mixture, after beingallowed to stand at 4° C. overnight, was centrifuged and again suspendedin 100 μl of NETBN solution 150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl (pH7.5), 0.1% BSA, 0.05% Non-idet (NP)-40!. To this suspension was addedthe above-treated mixture of labeled cell extract of ascites, and themixture was allowed to stand at 4° C. overnight. This mixture was thencentrifuged, and the resulting precipitate was suspended in 500 μl ofNETBN solution. This procedure was repeated 5 times to thereby removeunadsorbed labeled substance, and the pellets were resuspended with 50ml of electrophoresis sample buffer. The polyacrylamidegelelectrophoresis was performed in accordance with the method of Laemmli,U. K. Nature, 227, 680 (1970)!. After migration, the gel was immersed in50% trichloroacetic acid (TCA) for 1 hour and was washed four times withdistilled water for 30 minutes for each wash to thereby remove the TCA,after which the gel was immersed in dimethylsulfoxide (DMSO) for 1 hour.Thereafter, the gel was immersed in DMSO containing 10%2,5-diphenyloxazole (DPO) for 1 hour. After washing three times withdistilled water for 30 minutes for each wash, the gel was dried. Thedried gel was radioautographed, and the immnunoprecipitation pattern wasexamined. The radioautograms are shown in FIG. 2. From FIG. 2, themonoclonal antibodies MoAb12, MoAb52, MoAb78 and MoAb98 were found tocombine with hbFGF in cell extract.

Example 6

The antibody valencies of the 4 lines which showed immunoprecipitationin example 5, selected from the monoclonal antibodies described inExample 4, namely the monoclonal antibodies MoAb12, MoAb52, MoAb78 andMeAb 98 were determined by the limiting dilution method.

That is, each of the ascites fluids of monoclonal antibody MoAb12,MoAb52, MoAb78 or MoAb98 obtained in Example 4 was diluted with IHmedium containing 10% fetal bovine serum, and the quantity of antibodiesin the dilution was determined by the EIA method mentioned in Example 2(2). The results are shown in FIG. 3. In FIG. 3, - - - β - - - indicatesthe results for MoAb12, - - - □ - - - indicates the results forMoAb52, - - - ▪ - - - indicates the results for MoAb78, and - - - ∇- - -indicates the results for MoAb98.

FIG. 3 shows that the ascites containing the above antibody show alimiting dilution rate of more than 1×10⁶, that is, these 4 antibodiesare very high in antibody valency.

Example 7

(Determination of recognition site to the antigen)

Recognition sites of the four antibodies, which show high antibodyvalencies obtained in Example 6, to the antigen were determined bycompetitive analysis.

As competitors, hbFGF obtained in Reference Example 3, synthetic peptidePep 1: Pro-Ala-Leu-Pro-Glu-Asp-Gly-Gly-Ser-Thr (which is obtained byadding Tyr to the polypeptide of N-terminal amino acids Nos. 2 to 10,Regulatory Peptides, 10, 309-317(1985)), synthetic peptide Pep 2:Leu-Pro-Met-Ser-Ala-Lys-Ser (which corresponds to the amino acids Nos.142 to 147 obtained in Reference Example 17), bFGF mutein N14 (obtainedin Reference Example 12), and bFGF mutein N41 (obtained in ReferenceExample 15).

The synthetic peptides were adjusted to the concentration of 100 μg/mland diluted with IH medium (Iscov and Ham F12 mixed medium at a ratiowith 1:1) containing 10% FCS. The extract containing the mutein N14 orN41 obtained in Reference Example 12 (2) or 15 (2) was diluted with IHmedium containing 10% FCS. The antibodies, obtained in Example 4, werediluted to their binding titers showing between 0.7 and 1.0 withabsorbance at 415 nm. So, dilution factor about MoAb12, MoAb52 andMoAb98 was 5×10⁵, and about MoAb98 was 5×10⁴. In the above dilution, IHmedium containing 10% FCS was used as a diluent. To the diluted antibodysolution were added the diluted competitor. After stirring the mixture,the diluent was warmed at 37° C. for 30 minutes. The amount of theunbound antibody was measured by EIA method shown in Example 2 (2). Theresults in case of using synthetic peptide are shown in FIGS. 4 to 7.FIG. 4 shows the results on the monoclonal antibody MoAb12, FIG. 5 showsthe results on the monoclonal antibody MoAb52, FIG. 6 shows the resultson the monoclonal antibody MoAb 78, and FIG. 7 shows the results on themonoclonal antibody MoAb98.

In these figures, - - -  - - - denotes the results of hbFGF, - - -◯ - - - denotes the results of Pep 1, - - - ▪ - - - denotes the resultsof Pep 2, these being used as competitor, and the vertical axis denotesthe absorption at 415 mm.

As shown in the FIGS. 4 and 6, the monoclonal antibodies MoAb12 andMoAb78 are competitively inhibited to combine with bFGF and Pep 1. Thisindicates that the monoclonal antibodies MoAb12 and MoAb78 recognize theN-terminal amino acids of Nos. 2 to 10.

As shown in the FIG. 5 and FIG. 7, the monoclonal antibodies MoAb52,MoAb98 are not competitively inhibited to Pep 1 and Pep 2.

In order to determine the recognition site of the monoclonal antibodies,the competition analyses were conducted using the mutein N14 (obtainedin Reference Example (2) or N41 (obtained in Reference Example 15) bythe method described in the above, and the results are shown in FIGS. 8and 9. In these figures, - - -  - - - denotes the results ofhbFGF, - - - ∇ - - - denotes the results of the mutein N14, - - -◯ - - - denotes the results of the mutein N41, and the vertical axisdenotes the absorption at 415 mm, and the horizontal axis shows thetotal protein in the E. coli extract obtained in Reference Example 3.

As shown in FIG. 8 and FIG. 9, the monoclonal antibodies MoAb52 andMoAb98 are competitively inhibited to combine with mutein N14, but notwith mutein N41. This indicates that the monoclonal antibodies MoAb52and MoAb98 recognize the amino acids of Nos. 15 to 41.

The competitive analysis was carried out on bovine acidic FGF (baFGF)purchased from R & D Systems Inc., U.S.A.! and human bFGF obtained inReference Example 3 by the method described in the above, and theresults are shown in Table 5. The results are indicated by opticaldensity of the substrate by EIA method.

                  TABLE 5    ______________________________________           baFGF         hbFGF           10 μg/ml                  80 ng/ml   10 μg/ml                                      80 ng/ml    ______________________________________    MoAb12   1.049    0.975      0.396  1.077    MoAb52   0.997    0.923      0.230  0.722    MoAb78   0.978    0.962      0.062  0.523    MoAb98   0.948    0.921      0.095  1.356    ______________________________________

As shown in Table 5, the monoclonal antibodies MoAb12, MoAb52, MoAb78and MoAb98 do not cross-react with bovine bFGF.

All of the above results are shown in the following Table 6.

                  TABLE 6    ______________________________________             Monoclonal Antibody HbF             12   52          78     98    ______________________________________    hbFGF      +      +           +    +    baFGF      -      -           -    -    N14        -      +           -    +    N41        -      -           -    -    Pep1       +      -           +    -    Pep2       -      -           -    -    ______________________________________

In the Table 6, + denotes that they are competitive, and - denotes thatthey are not competitive.

Example 8

(Purification of the monoclonal antibody from ascites)

The monoclonal antibody MoAb12, MoAb52, MoAb78 or MoAb98 was inoculatedto 10 mice, and 20 to 30 ml of ascites were collected. The ascites weresubjected to centrifugation at 2,000 rpm (Hitachi refrigratedcentrifuge) to remove cells, and further subjected to centrifugationwith Spinco SW 28 roter (Beckman, U.S.A.) at 4° C. for 2 hours to removethe insoluble proteins and fats. To the supernatant thus obtained thereis added ammonium sulfate so as to 40% saturation, and the mixture wasstirred gently in ice bath for 1 hour.

The precipitate was subjected to centrifugation with Sorval SS34 roter(Dupont, U.S.A.) at 4° C., 15,000 rpm. The pellet was dissolved inBuffer 1 20mM Tris.HCl (pH 7.9), 40 mM NaCl! so that the proteinconcentration is 10 to 15 mg/ml. Thus obtained solution is dialyzed forthe Buffer 1 at 4° C. overnight. Thus obtained solution is passedthrough the column of DEAE-cellulose (DE-52, Whatman, U.S.A.) to adsorb.The elution was carried out employing the linear gradient from Buffer 1to 0.4M NaCl.

The immunoglobulin fractions were recovered. To the fraction were added40% saturated ammonium sulfate so as to emerge precipitate. Theprecipitate was dissolved in Buffer 2 (0.1M NaHCO₃) so as to the proteinconcentration being 10 to 20 mg/ml, and the solution was subjected todialysis to Buffer 2 at 4° C. for two overnights. The Buffer 2 werereplaced every day.

Furthermore, the dialyzate is subjected to hydroxy apatite column (HCAcolumn). As the initiation buffer, 10 mM sodium phosphate buffer (pH6.8) was used, and as the elution buffer, 5mM sodium phosphate buffer(pH 6.8) was used. The elution was carried out by linear gradientelution from the initial buffer to the elution buffer. Thus obtainedeluate containing the antibody was preserved at 4° C.

Example 9

(Purification of hbFGF by antibody column)

5 ml of Affi-Gel-10 (Bio-Rad, U.S.A.) was put on sintered filter, waswashed with ten volumes of ice-cooled isopropanol and with ten times ofice-cooled distilled water. Thus obtained gel is transfered to areaction vessel. To the gel was mixed with 15 mg of monoclonal antibodyMoAb78 in the volume of 5 to 15 ml (dissolved in Buffer 2 or phosphatebuffer, in Example 8) to react at 4° C. overnight.

Monoethanol amine (pH 8.0) was added to the reaction mixture with theconcentration of 0.01M, and leave at room temperature for one hour toinactivate the unreacted site. The gel was washed with 10 times (volume)of Buffer 2 (Example 8). 2 ml of the gel was packed into a column, andthe column was equilibrated with the initiation buffer 20mM Tris.HCl (pH7.6), 1 mM EDTA, 0.15M NaCl, 0.05% NP-40!.

On the other hand, the extracts of a transformant Escherichia coliDH1/pTB744 (the extracts containing mutein CS4), is diluted three timesby the addition of the initial buffer. The diluent was applied to saidcolumn at a flow rate of 20 ml/hour to adsorb the mutein CS4 to theantibody. After the adsorption, the column was washed with 20 ml of theinitial buffer, and then the elution was carried out by using 20 ml ofhigh salt buffer 20 mM Tris.HCl (pH 7.6), 1 mM EDTA, 1M NaCl, 0.05%NP-40!, 20 ml of an elution buffer A 0.2M acetate buffer (pH 4.5), 0.2MNaCl!, 20 ml of an elution buffer B 0.2M acetic acid (pH 2.5), 0.2MNaCl! in that order, wherein the flow rate is 20 ml/hour and thetemperature is 4° C. Thus obtained fractions were subjected toelectrophoresis in accordance with the method described in Laemmli,Nature 277, 680 (1970) employing 17.25% acrylamide. Proteins weredetected by silver staining.

The results are shown in FIG. 10. In FIG. 10, MK denotes the result ofmolecular marker, A denotes that of the crude extract, B denotes that offlow through fractions, C denotes that of the eluent of the high saltbuffer, D denotes that of the elution buffer A, and E denotes that ofthe elution buffer B. Just after the elution, the pH of D fraction and Efraction was adjusted to pH 7.5 by adding 1M Tris.HCl (pH 9.5). The FGFactivity on the fractions were measured in accordance with the method ofReference Example 5 (3). The results are shown in Table 7. In Table 7, Ato E denote the same as above.

                  TABLE 7    ______________________________________    Protein (A) (μg)                    FGF Activity* (B) (μg)                                   B/A    ______________________________________    A     63000         96             0.0015    B     63000         19             0.0003    C     28            0.003          0.0011    D     17            0.049          0.0030    E     33            23             0.69    ______________________________________     Note: *: The FGF activity is shown in equivalent amount of the bovine     pituitary derived bFGF (perchared from Takara Shuzo, Japan) on the basis     of incorporation of .sup.3 Hthymidine.

Example 10

(Measurement of hbFGF mutein by EIA method using monoclonal antibody)

(1) The antibody MoAb78 obtained in Example 4 was subjected topurification from ascites fluid in accordance with the method of Example8. Thus obtained antibody was concentrated to more than 2 mg/ml, andsubjected to dialysis in 0.2M sodium phosphate buffer (pH 7.0). To thusobtained 1.4 ml solution of monoclonal antibody MoAb78 (concentration2.8 mg/ml), 50 μl of S-acetylmercaptosuccinic anhydride (Aldrich Co.,U.S.A.) dissolved in N,N'-dimethylformamide was added so as to reach theconcentration of 11.5 mg/ml. The air in the reaction vessel is replacedby nitrogen gas. The vessel was sealed, and subjected to stirring so asto cause the reaction of introducing SH group. The unreactedS-acetylmercaptosuccinic acid anhydride was inactivated by the treatmentfor 10 minutes with 130 μl of 0.2M Tris.HCl (pH 7.0), 13 μl of 0.2M EDTAand 130 μl of 2M hydroxyamine (pH 7.0). The MoAb78 was recovered by gelfiltration using a column packed with Sephadex G-25 (diameter 1 cm×80cm, Pharmacia, Sweden) (flow rate: 20 ml/hour).

(2) 10 mg of horse radish peroxidase (HRP, Behringer Manheim, Grade I,West Germany) was dissolved in 1.4 ml of 0.1M phosphate buffer (pH 6.8).On the other hand, 14 mg of N-hydroxysuccinimide ester of N-(4-corboxycyclohexyl methyl)maleimide was dissolved in 335 μl of DMF, and 100 μlof thus obtained solution was added to the HRP solution above mentioned.The air in the reaction vessel was replaced by nitrogen gas, and thevessel was sealed. After 1 hour reaction at room temperature, proteinsof the portion of HRP introduced with maleimide group were recovered bygel filtration using a colomn packed with Sephadex G-25 as in the above(f).

(3) 6 ml of the portion of the monoclonal antibody MoAb78 introducedwith SH group obtained in the above (1) and 2 ml of the portion of HRPintroduced with maleimide group obtained in the above (2) were mixed,and the mixture was concentrated to 1 ml using collodion bag (Sartorius,West Germany) under reduced pressure at 4° C. for 20 hours. After thereaction, the unreacted HRP introduced with SH group was removed withthe use of Ultrogel AcA44 (LKB Co., diameter 1 cm×80 cm, Sweden) column(flow rate: 10 ml/hour). In the eluates, the fraction containing 2.4HRP/antibody has the most high HRP number per antibody molecule. Theproduct thus obtained was employed in the EIA in the following item (4).

(4) The monoclonal antibody MoAb52 was purified by the manner describedin the above (1). The monoclonal antibody MoAb52 was diluted with PBS soas to be 10 μg/ml or 20 μg/ml, and the diluent was poured intoImmunoplate (Nunc, Denmark) so as to be 100 μl/well. The plate was keptstanding at 4° C. overnight to adsorb the monoclonal antibody MoAb52 tothe plate. After removing the antibody which is not adsorbed, the platewas washed with PBS thrice, PBS containing 0.01% merthiolate and 1%bovine serum albumin (BSA) was added to the plate at 200 μl/well, andthe plate was kept standing at 4° C. overnight.

(5) The cell extract containing bFGF mutein C86 obtained in ReferenceExample 3 was diluted with PBS containing 0.1% BSA. From the plateobtained in the above (4), BSA solution was removed, the plate waswashed with PBS four times, and the diluted bFGF mutein C86 was added tothe plate so as to be 100 μl/well to adsorb to the plate at 4° C.overnight. The unreacted mutein C86 was removed, and the plate waswashed with PBS four times. The monoclonal antibody conjugated with HRP(HRP-MoAb78) obtained in the above (3) was diluted with PBS containing0.1% BSA to 1/300, and the diluent was added to the plate so as to be100 μl/well. The reaction was carried out for 4 hours at roomtemperature. After removing the antibody, the plate was washed with PBSfor 6 times, substrate for oxidase (Bio. Rad Co. U.S.A) was added to theplate so as to be 100 μl/well. Quantification was accomplished byabsorbance measurements at 415 nm, and it was confirmed that a smallamount of the mutein C86 was produced.

(6) In FIG. 11, the detection curve is shown in case that the amount ofmonoclonal antibody MoAb52 which is fixed to the plate is 1 μg/well(- - - ◯ - - - ), and 2 μg/well (- - -  - - - ). The horizontal axisshows the concentration of bFGF added, and the vertical axis shows theabsorbance at 415 nm of the solution caused by HRP-MoAb78.

From the FIG. 11, it is taught that the concentration of 0.5 ng/ml ofbFGF can be detected, when the monoclonal antibody MoAb52 is adsorbed tothe plate in an amount of 2 μg/well.

(7) The monoclonal antibody MoAb98 was adsorbed to the plate in anamount of 2 μg/well, according to the method of the above (4), and themeasurement of absorbance at 415 nm was carried out in accordance withthe method of the above (5). The results are shown in FIG. 12. Thehorizontal and vertical axes show the same as those of FIG. 11. FromFIG. 12, it is taught that at least 0.5 ng/ml of bFGF can be detected byusing the monoclonal antibody MoAb98.

Example 11

(The measurement of hbFGF mutein by EIA method employing monoclonalantibody)

The cell extract containing human bFGF mutein C86 obtained in ReferenceExample 13 was treated with the manner of Example 9 to measure theexpression amount of the mutein. The results indicate that the muteinC86 is expressed in the cell in a slight amount.

Example 12

(The measurement of hbFGF mutein by EIA method employing monoclonalantibody)

The cell extract containing human bFGF mutein C129 obtained in ReferenceExample 14 was treated with the manner of Example 9 to measure theexpression amount of the mutein. The results indicate that the muteinC129 is expressed in the cell in a slight amount.

Example 13

(Detection of hbFGF by the method of Western blotting)

hbFGF obtained in Reference Example 3 was subjected to electrophoresisemploying 17.25% acrylamide gel Laemmli, Nature, 277, 680-685 (1970)!,and it was transferred Journal of Biochemical and Biophysical Methods,10, 203-209 (1984)! on the membrane of nitrocellulose by using Sartoblot(Sartorius, West Germany). This membrane was washed with TBS (20 mMTris.HCl (pH 7.5), 0.5M NaCl! for 5 minutes twice, and kept standing inTBS containing 4% BSA at room temperature for one hour to block theunreacted material on the membrane. Thus obtained membrane was washedwith TBS containing 0.05% Tween 20 (TTBS) for 5 minutes twice.

The monoclonal antibody MoAb12 or MoAb78 was diluted with TTBScontaining 1% gelatin so as to 1/3000. To thus obtained dilution saidnitrocellulose membrane was inserted, and reaction was carried outovernight. After the reaction, the reaction liquid was removed, and themembrane was washed with TTBS for 5 minutes twice.

The secondary antibody, i.e., anti mouse IgG goat serum (Bio Rad,U.S.A.) labeled with HRP, was diluted to 1/3000 by TTBS containing 1%gelatin.

To the membrane obtained above was added the diluted secondary antibody,and reaction was carried out at room temperature for one hour. Themembrane thus obtained was washed with TTBS for 5 minutes thrice, andthen washed with TBS for 5 minutes twice. After that, to the membranewas added 0.05% 4-chloro-1-naphtol as substrate and 0.015% hydrogenperoxide, and the reaction was carried out for 15 minutes.

In FIG. 13, the results of Western blotting in case the monoclonalantibody MoAb78 was used as primary antibody. The lane 1 shows theresults of 1 μg of bFGF, the lane 2 shows the results of 300 μg of bFGF,the lane 3 shows the results of 100 μg of bFGF, wherein bFGF waselectrophoresised and transferred. M shows a marker, and the numerals invertical axis show molecular weight. bFGF was detected in the samesensitivity when the monoclonal antibody MoAb12 instead of MoAb78.

The following references, which are referred to for their disclosures atvarious points in this application, are incorporated herein byreference.

Nature, 249, 123 (1974)

National Cancer Institute Monograph, 48, 109(1978)

Proceedings of the National Academy of Sciences, U.S.A., 82, 6507(1985)

European Patent Publication No.237,966

European Molecular Biology Organization (EMBO) Journal, 5, 2523(1986)

Genetic Enginnering, Academic Press (1983), pp.31-50

Genetic Enginnering: Principles and Methods, Prenum Press (1981), vol.3,pp.1-32

Journal of Inmunological Methods, 80, 55(1985)

Nature, 256, 495(1975)

Journal of American Medical Association, 199, 549(1967)

Proc. Natl. Acad. Sci. U.S.A., 80, 3513-3516(1983)

Molecular and Cellular Biology, 3, 280(1983)

Nucleic Acids Research 1, 1513(1979)

Proc. Natl. Acad. Sci. U.S.A., 72, 3961(1975)

Nucleic Acids Research, 9, 309(1981)

Nucleic Acids Research, 11, 3077-3085(1983)

Molecular Cloning (1982), A Laboratory Manual, Cold Spring HarborLaboratory, U.S.A.

Methods in Enzymology, 101, 20-78(1983)

Nature, 227, 680(1970)

Regulatory Peptides, 10, 309-317(1985)

Journal of Biochemical and Biophysical Methods, 10, 203-209(1984)

What we claim is:
 1. A monoclonal antibody which specifically binds withbasic fibroblast growth factor, wherein the antibody has the followingcharacteristics:(a) it has a molecular weight of about 140 to 160kilodaltons, (b) it does not cross-react with acidic fibroblast growthfactor, (c) it belongs to the immunoglobulin class IgM or IgG, and (d)it is capable of detecting human basic fibroblast growth factor in aconcentration of at least 0.5 ng/ml of basic fibroblast factor by EIAmethod.
 2. The monoclonal antibody as claimed in claim 1, wherein thebasic fibroblast growth factor bound to is a human fibroblast growthfactor containing the amino acidsequence:Phe-Phe-Leu-Arg-Ile-His-Pro-Asp-Gly-Arg-Val-Asp-Gly-Val-Arg-Glu-Lys-Ser-Asp-Pro.3. A cloned hybridoma comprising a splenic cell from a mammal immunizedwith human basic fibroblast growth factor and a homogenic or heterogeniclymphoid cell, wherein the hybridoma is capable of producing amonoclonal antibody which specifically binds with basic fibroblastgrowth factor, wherein the antibody has the followingcharacteristics:(a) it has a molecular weight of about 140 to 160kilodaltons, (b) it does not cross-react with acidic fibroblast growthfactor, (c) it belongs to the immunoglobulin class IgM or IgG, and (d)it is capable of detecting bFGF in the concentration of at least 0.5ng/ml of basic fibroblast growth factor by EIA method.
 4. The clonedhybridoma as claimed in claim 3, wherein the basic fibroblast growthfactor is a human fibroblast growth factor containing the amino acidsequence:Phe-Phe-Leu-Arg-Ile-His-Pro-Asp-Gly-Arg-Val-Asp-Gly-Val-Arg-Glu-Lys-Ser-Asp-Pro.